SOME CONSEQUENCES OF THE BINDING OF AFLATOX1N B1 WITH PLASMA MEMBRANE ON THE REGULATION OF INTRACELLULAR Ca2+ HOMEOSTASIS. BY s ; I ADERONKE OLANREWAJU ADEBAYO B.Sc.(Hons), M.Sc. Biochem., University of Ibadan. A thesis in the Department of Biochemistry Submitted to the Faculty of Basic Medical Sciences in partial fulfilment of requirements for the degree of DOCTOR OF PHILOSOPHY of the University of Ibadan March, 1992 UNIVERSITY OF IBADAN LIBRARY - 2 ABSTRACT The possible influence of aflatoxin a potent hepatocellular carcinogen on the regulation of intracellular Ca^+ homeostasis has been studied using the red cell as a model. Preliminary work on the interaction of the toxin with the red cell membrane using spectro- fluometric analysis indicated that the toxin binds spontaneously and irreversibly to the red cell membrane. The binding is highest at pH 4 and least at pH 10. Results obtained from studies using equilibrum dialysis technique show that about 4 \nmoles of the toxin bind to one microgram membrane protein. Although the exact membrane component to which aflatoxin Bi binds is not known, experiments carried out to determine the influence of aflatoxin B^ on the activity of the calcium pumping protein revealed that the toxin inhibited the calmodulin-stimulated erythrocyte membrane Ca 2+-ATPase activity by about 50 percent, while it has little or no effect on its basal activity. Kinetic analysis of the UNIVERSITY OF IBADAN LIBRARY 3 inhibition shows that, the toxin reduces the Vmax and Km of the calmodulin-stimulated enzyme by 50 percent in a non-competitive manner, On the other hand, the carcinogen had no significant influence on the kinetic parameters of the enzyme in the non-activated state. Similar results were obtained for the triton X-100 solubilized and calmodulin affinity chromato­ graphed enzyme. In this instance aflatoxin 3-j inhibited the calmodulin-stimulated purified enzyme by 50 percent with or without preincubation on ice for half an hour. Again, the toxin had little or no effect on the basal activity of the enzyme in the absence of calmodulin. Analysis of the results obtained using varying concentrations of ATP shows that the Km and Vmax of the non-activated enzyme were not altered by the toxin while both the Vmax and Km values were reduced by about 50 percent in the presence of calmodulin*, In addition aflatoxin B-j inhibited Diphosphotidyl glycerol (cardiolipin) by about 28>S while it has no effect on the basal activity of the enzyme. Although, UNIVERSITY OF IBADAN LIBRARY 4 the inhibition of the membrane bound or purified Ca o + ATPase by the toxin is concentration dependent, varying concentrations of phosphatidyl serine and phosphatidyl choline do not affect the inhibition of the purified enzyme by afla toxin B-j. Results obtained with triton X-100 solubilized enzyme shows that triton X-100 alone could not activate the enzyme. Thus at triton X-100: protein ratio of 2, the enzyme was stimulated by calmodulin. This activity was sensitive to inhibition by the toxin. In this instance, the calmodulin-stimulated activity was inhibited by about 50%, while at lower ratios of the triton X-100 to protein there was no significant inhibition of enzyme. Results of experiments carried out on the 12i+KDa fragment, which was produced as a result of exposure to calpain a Ca2+-dependent cysteinj^protease, indicated that the toxin has no effect whatsoever on the activity of the fragmented enzyme, Similarly experiments on limited proteolysis of the Ca2+ ATPase by trypsin to give the 90KDa fragment which still retains its calmodulin binding UNIVERSITY OF IBADAN LIBRARY - 5 - domain and the 76KDa fragment which has lost its calmodulin binding domain revealed that the aflatoxin inhibited the 90KDa fragment by about 50% while the 76KDa fragment is not affected at all. Altogether, -these findings show that aflatoxin B^ inhibits the plasma membrane Ca 2 pumping ATPase by interacting with the enzyme at the calmodulin binding domain. The nature of the exact amino acid residue to which the toxin binds is however not known. The implication of these observations is that Ca^+ extrusion may be hampered in situations where the cell is poisoned by the aflatoxin UNIVERSITY OF IBADAN LIBRARY 5 ACKNOWLEDGEMENTS I will like to first of all thank my father, my God, the King of Kings and the Lord of Lords, for His abundant life, His mercies, grace and Love that saw me throughout my entire programme. I wish to express my appreciation to my supervisors Prof.O.O. Olorunscgo and Prof. A.O. Uwaifo. I wish to particularly thank Prof.O.O. Olorunsogc- for providing part of the cherr.5 -als used during the period of this work. I am very grateful for his guidance, support and for arranging my trip to Switzerland. I am most grateful for his endearment when I was bereaved. Also, for his understanding, encouragement and honest criticisms at each stage of the project and write up. I also thank him for meticulously going through the write up and proof reading the manuscripts. Many thanks to Prof. Enitan Abisogun Bababunmi, Godwin 0. Emerole and Maduagwu and also Drs Fafunso, Faparusi, Jeyakumar, Nwankwo, my senior colleagues UNIVERSITY OF IBADAN LIBRARY in the department for their various kind assistance,encouragement and support. I am very grateful to Mr. E.O. Farombi for upholding me in prayers especially during my difficult times and very, very grateful to Dr. Gbolahan Waheed Okunade and his family for taking me like a sister and for his kind gesture and always willing to help at odd times. I also thank Dr. (Kiss) Ronke Csowole for her kind assistance. I am especially very grateful to Prof. Ernesto Carafoli of Swiss Federal Institute of Technology, Zurich, for allowing me to come and do part of my research in his Laboratory. I also thank the International Union of Biochemistry for awarding the fellowship. Many thanks to Dr. Paolo Gazzoti, for his kind gesture, for introducing me into the different techniques that I used during my stay there. I also want to express my gratitute to Drs Felix Kessler, Hans Rhyner, Rocco Falchetto, Matin Pruschy, Emanual Strehler and Joachim Krebs for their kind assistance and support. UNIVERSITY OF IBADAN LIBRARY 8 I wish to thank Wole Owojuyigbe, Gbenga Adenuga, and Mrs Adenike Ibitayo (Nee Fasogbon) for their cooperation, and encouragement during the period of the examination. My sincere thanks to my friends, Mrs. Ronke Mosuro (Nee Idowu) and family for accommodating me during my most difficult time, Mrs. Simi Okunaiya (Nee Kuku), Mrs Bisi Olagunju (Nee Ogunsekan) and families for their encouragement and kind assistance throughout my programme. I also thank Miss 'Detola Olawore for her encouragement and assistance. Many thanks to my late husband's family, the Fajinmi's especially the senior brother Mr. Sunday Fajinmi for his concern, kind assistance and support. I wish to thank the Christian family fellowship, U.I. and Yeshua Bible School, for upholding me in their prayers throughout my programme. May thanks to the ab.dt. partnership for drawing the graphs and for their support especially during my various trips to Lagos. My sincere thanks to my parents, brothers and sisters for their concern, encouragement, moral support, understanding and most importantly UNIVERSITY OF IBADAN LIBRARY - 9 - for their prayers. I wish to express my unreserved gratitude to my loving son Mr. Babatunde Adeboye (Jnr) for his understanding and for his patience whenever I was working late in the laboratory. He is a source of joy to me. Finally, I wish to thank Mr. Michael Segilola for typing the thesis. I am very grateful to all. Aderonke 0. Adebayo UNIVERSITY OF IBADAN LIBRARY 10 - CERTIFICATION We certify that this work was carried out by Miss Aderonke Olanrewqju Adebayo in the Department of Biochemistry, University of Ibadan, Ibadan, Nigeria. Supervisor Supervisor Prof. Anthony 0. Uwaifo P-F°£* Olufunso 0. Olorunsogo UNIVERSITY OF IBADAN LIBRARY 11 TABLE OF CONTENTS page Abstract ................................... 2 Acknowledgements ........................... 6 Certification ................................ 10 Table of Contents .......................... 11 List of Plates ...................... 16 List of Figures ...................... 17 List of Tables .... 21 Abbreviations ................................ 23 CHAPTER ONE: INTRODUCTION ................ 1:1 Control of Intracellular Calcium...... 25 1:2 Discovery and general properties 1 of Ca^- pump ........................ 28 A 1:3 Primary structure of the Ca^+- translocating ATPase .............. 38 1:4 Secondary structure of the plasma membrane Ca^+ ATPase .............. 52 1:5 Calcium pump isoforms ............... 60 1:6 Mechanism of the Ca^+ pump .......... 70 1:7 Modes of activation of plasma membrane Ca^+ pump ................. 75 UNIVERSITY OF IBADAN LIBRARY Page 1 :8 Modulation of Ca — ATPase by calmodulin ......................... 73 1:9 Activation of Ca 2~ pump by lipids .. g7 1:10 Activation of the purified Ca' — ATPase of the erythrocyte membrane by controlled proteolysis ......... 91 1:11 Modulation of Ca^t-ATPase by calpain 99 1:12 Activation of Ca^— pump by kinase mediated phosphorylation ........... io3 1:13 Activation by oligomerisation ....... 109 1:14- Historical Background ............... 1:15 Chemistry of aflatoxins ............. ^jjr' 1:16 Carcinogenesis of aflatoxin ......... '• * 1:17 Occurrence of aflatoxin in some Nigerians foods ................ 121 1:18 Inhibition of DNA synthesis by aflatoxin ............... 122 1:19 Inhibition of protein synthesis by aflatoxin B«j ........................ 123 1:20 Alterations of RNA metabolism by aflatoxin B^ ........................ 125 UNIVERSITY OF IBADAN LIBRARY - 13 - Fage 1:21 Effect of aflatoxin B-j on nucleolar morphology ........................... 126 1;22 Interaction -with proteins ............ \21 1:23 Interactions of aflatoxins with other cellular compounds .................. 128 1:24 Activation and Toxicity of aflatoxin.. ^30 1:25 Properties of aflatoxins ............. 134 1:26 Objective ............................ 137 CHAPTER TWO: MATERIALS AND METHODS ........ 2:1 Collection of samples ................ 44* 2:2 Preparation of erythrocyte ghost membranes ............................ 141 2:3 Determination of protein ............. 2:4 Measurement of protein concentration of purified Ca^— ATPase enzyme samples 151 2:5 Assay of membrane-bound ( C a + Mg +)- ATPase ............................... 153 2:6 Purificationp of erythrocyte plasma membrane Ca^+-ATPase on calmodulin affinity column........................ 159 2:7 Purification of the enzyme in the absence of added phosphatidyl choline.. 170 UNIVERSITY OF IBADAN LIBRARY 14 Page 2:8 Assay of purified C er+- ATPase ...... ^J2 2:9 Binding studies on the interaction of aflatoxin B/j to erythrocyte membrane ........................... 174 2:10 Proteolysis of Ca^+-ATPase ......... 178 2:11 Electrophoretic separation of membrane-bound and purified proteins on continuous gradients of sodium dodecyl sulphate-polyacrylamide gels 180 2:12 Silver staining technique .......... -93 CHAPTER THREE: EXPERIMENTS AND RESULTS Experiment 1: Investigation of the binding of aflatoxin to erythro­ cyte membrane ........... 203 Experiment 2: Influence of aflatoxin B^ on erythrocyte membrane C e r+- ATPase activity ............ 211 Experiment 3: Inhibition of erythrocyte purified Ca^+-ATPase by aflatoxin B^ ............... 221 V: 1 **•••• UNIVERSITY OF IBADAN LIBRARY 15 Page Experiment 4: Effect of triton X-100 and cardiolipin on the inhibition of Ca^+-ATPase by aflatoxin ............ 231 Experiment 5: Interaction of aflatoxin B with partially proteolysed purified Ca^+-ATPase.... . 243 CHAPTER FOUR: DISCUSSION.................. 251 SUMMARY OF RESULTS ......... 276 CONTRIBUTION TO KNOWLEDGE.................. 279 REFERENCES ................. 280 APPENDIX ................... 347 UNIVERSITY OF IBADAN LIBRARY 16 - LIST OF PLATES Page 1. SDS-polyacrylamide gel electropho- resis of erythrocyte purified AC a +- pumping ATPase ............ . 196 2. SDS-polyacrylamide gel electropho­ resis of erythrocyte ghost membranes.... 197 3. SDS-polyacrylamide gel electrophoresis of partially trypsinised purified Ca^+-pumping ATPase ............ . 247 UNIVERSITY OF IBADAN LIBRARY 17 - LIST OF FIGURES y Fig.l: The Denny-Jaffe reagent .......... 41 2: The complete primary structure of plasma membrane Ca^2 -ATPase............ 43 3: Proposed model for the overall topology of plasma membrane Ca - ATPase (PMC As )...................... 46 4a: Hypothetical scheme of the function pf the calmodulin binding domain in the plasma membrane Ca2+-pump.............. 54 4b: A model of the interaction of calmodulin with the plasma membrane C— a 2+-pump ............................ 53 5: Structural diagram of the Ca2+-ATPase molecule....... 56 6: Domain assignment in the plasma membrane calcium pump and location of isoform variable regions .......... 65 7: Scheme for (Ca2++ Mg2+)-ATPase transport cycle ...................... 72 8 : Amino acid sequence of calmodulin ..... 95 UNIVERSITY OF IBADAN LIBRARY 18 - Page Fig. 9: Scheme for trypsin proteolysis of purified Ca^+-pump in the presence of different effectors... 95 10: Location of NH2 and COOH termini of tryptic fragments of molecular masses 90, 85 and 76KDa ........................... 96 11: Domain structure of calpain ...... 101 12: Model of the erythrocyte membrane calcium pump............ 105 13: Structure of aflatoxin B-̂ ....... 1^5 14: Structures of the different types of aflatoxin 117 15: The metabolic activation of aflatoxin by rat liver ........ 132 16: Schatehard plot of the binding of aflatoxin Bi to erythrocyte membranes 209 17: Effect of varying concentrations of aflatoxin on the basal and calmodulin-stimulated activity of erythrocyte plasma Ca^+-ATPase .... 215 UNIVERSITY OF IBADAN LIBRARY - 19 - Page Fig. 18: Inhibition of calmodulin- stimulated erythrocyte membrane Ca2+-ATPase by aflatoxin B-j........... 216 19: Influence of aflatoxin B-j on ATP- dependence of erythrocyte membrane Ca2+-ATPase ........................ 217 20: Influence of aflatoxin B^ on the ATP dependence of calmodulin- stimulated erythrocyte membrane Ca2+-ATPase ........................ 218 21: Effect of varying concentrations of aflatoxin B^ on the basal and calmodulin-stimulated activity of the purified ATPase ................. 224 22: Inhibition of the calmodulin- stimulated purified enzyme by aflatoxin B ^ ..................... 226 23: Influence of aflatoxin B^ on the ATP dependence of erythrocyte purified ATPase in the absence of calmodulin ................. 227 24: Influence of aflatoxin 3^ on the ATP-dependence of the calmodulin- stimulated purified ATPase ........ 229 UNIVERSITY OF I ADAN LIBRARY 20 Page Fig. 25: Effect of varying concentrations of aflatoxin on cardiolipin - stimulated Ca^+-ATPase .......... . 236 26: Percentage inhibition of the cardiolipin-stimulated Ca^+- ATPase by aflatoxin ........... . 237 27: Influence of varying triton X-100: protein ratios on the inhibition of the Ca^+-ATPase by aflatoxin B! ................................ 238 28: Effect of varying concentrations of phosphatidyl choline on the inhibition of the Ca^+-ATPase by aflatoxin B-j ....................... 240 29: Effect of varying concentrations of phosphatidyl serine on the inhibition of the Ca^+-ATPase by aflatoxin .................... 242 UNIVERSITY OF IBADAN LIBRARY 21 - LIST OF TABLES Page Table 1: Calcium-dependent cellular reactions and processes .......... 26 2: Transporting systems in biological membrane .......................... 29 3: Calcium pumps in the plasma membrane of eukaryotic cells ..... 31 4: Properties of the plasma membrane Ca^+- A'TPase ...................... 36 2 5 : Ca^+-pump sequences showing similarities to mammalian calmodu­ lin ............................... 48 6 : Isoforms of the plasma membrane Ca *-pump ......................... 52 7 : Calmodulin-binding domain of various isoforms of the Ca^+-ATPase 69 8 : Effects of various modulators on the plasma membrane Ca^+-ATPase.... 76 9. Representative organisms from which calmodulin has been isolated.. 80 10: Calmodulin mediated enzymes and processes.......................... 84 UNIVERSITY OF IBADAN LIBRARY 22 Page Table 11: Local (Nigerian) foodstuffs known to support aflatoxin production.... ......... 113 12: Physico-chemical properties of the aflatoxins .......... 135 13: Protocol for protein estimation...... 14: Preparation of running and stacking gels (A) .................. 190 15: Preparation of running and stacking mini gels thin spacers ..... 194 16: Effect of pH and temperature on the binding of aflatoxin B^ to the erythrocyte ghost membranes ..... 207 17: Effect of aflatoxin on the Vmax and Km values of the erythrocyte plasma membrane Ca 2+-ATPase ......... 220 18: The effect of aflatoxin B^ on Vmax and Km values of the purified Ca^+- ATPase .................... . 230 19: Effect of aflatoxin B^ on the activity of calpanised Ca^+-ATPase..• 248 20: Effect of aflatoxin B-* on the activity of partially proteolysed Ca^+-ATPase ............. . 250 UNIVERSITY OF IBADAN LIBRARY 23 ABBREVIATIONS ATP Adenosine 5* triphosphate ATPase Adenosine 5' trisphosphatase BSA Bovine serum albumin CaM Calmodulin cAMP 3 ’ 5 ’-cyclic adenosine-5U monophosphate Ca2+-ATPase Mg -dependent Ca^ -pumping Adenosine triphosphatase DTT Dithicthreitol EDTA ethylene diamine tetracetic acid IOVs Inside-out vesicles Fig Figure g gram hPMCA human plasma membrane Ca^+-ATPase HEPES 4-(2-hydroxyethy1)-piperazine ethanesulphonic acid hr hour Km Michaelis constant M Molar mg milligramme Kr Molecular weight nMoles nanomoles ND not determined UNIVERSITY OF IBADAN LIBRARY pc phosphatidyl choline pH a measure of hydrogen ion concentration PMSF phenylmethylsulfonylfluoride PS Phosphatidyl serine rpm revolution per minute rPMCA rat Plasma Membrane Ca^+ ATPase SDS Sodium dodecyl sulphate SDS-PAGE Sodium dodecyl sulphate polyacrylamide TCA Trichloroacetic acid TEP1ED N,N,N',N'-tetramethyl-p-phenylenylene diamine Tris-HCl Tris (hydroxymethyl) amino methane hydrochloride ug microgramme umole micromole uM micromolar ^max maximal velocity of enzymatic reaction. UNIVERSITY OF IBADAN LIBRARY - 25 - CHAPTER ONE INTRODUCTION 1s1 The Control of Intracellular Calcium The recognition of the importance of calcium in cell functions dates back to the pioneering work of Ringer (1882, 1883) who established that calcium ions are important in the contraction of frog heart muscle. The work was later confirmed and extended by Locke (1894). Research activity in this area remained dormant until 1947 when Heilbrunn and Wiercinski showed that, the injection of a small amount of Ca^+ into a muscle fibre causes it to contract. Calcium ion is now well known to mediate a wide variety of cellular responses and processes such as cell motility, endo-and exocytosis and more complicated processes such as cell prolifera­ tion, fertilization and hormone secretion (Table l). It is now generally accepted that Ca^+ is a very important and possibly the most important second messenger in living cells. It has also been suggested that . Ca^+ may play the role of a primary messenger since it, directly interferes with UNIVERSITY OF IBADAN LIBRARY 26 TABLE 1 Calcium-dependent cellular reactions and processes. Enzyme/process Reference Phosphorylase kinase Cohen, Burchell and (glycogenolysis) Foulkes, 1978* Phospholipase A2 Wong and Cheung, 1979. Myosin light chain Walsh, Cavadore, Vallet Kinase and Demaille 1980. Erythrocyte Ca +-ATPase Schatzmann 1966 Adenylate cyclase Bronstom, Bronstom and Wolff 1977. Cell motility Tash and Mann 1973 Muscle contraction Ebashi 1958. Exo and endocytosis Linden, Dedman, Chafouleas, Means and Roth, 1981. Cell division and Welsh; Dedman, Brinkley, proliferation Means 1978. Fertilization Epel, Patton, Wallace and Cheung 1981 UNIVERSITY OF IBADAN LIBRARY - 27 - the generation of signals at the level of plasma membranes by regulating K+, Na+ and even Ca2+ currents (Carafoli and Crompton, 1978). An important consequence of the messenger role of Ca^+ is, the necessity for its precise regulation. Thus the control of the intracellular level of Ca^+ is also an essential step in metabolic regula- tion. The steady state concentration of Ca^* is about O 10 to 10 —9'M in mammalian cells, a value that is three or four orders of magnitude lower than the free Ca^+ concentration in the extracellular environment estimated to be about 1.5mM (Rasmussen and Goodman, 1977; Carafoli and Crompton, 1978). Maintenance of this concentration difference depends on the extrusion of the cation through the plasma membranes to balance its continous passive influx, sequestration within subcellular organelles such as mitochondria and sarcoplasmic reticulum and binding of Ca* to non-membranous ligands in cytosol. However, in erythrocytes, the low intracellular CaO^+ concentration is regulated mainly by the presence of membrane-bound pump that UNIVERSITY OF IBADAN LIBRARY 28 - drives calcium out of the cell against the / concentration gradient. Thus Cap^+ is transported across biological membrane by four basic mechanisms as shown in Table 2l. In most eukaryotic cells, the same membrane system may transport Ca^+ by more than one of these mechanisms (Carafoli, 1984). Na+/Ca^+ exchangers have been reported in plasma membrane and the inner mitochondrial membrane. 1.2 Discovery and general properties of Ca^* pump The existence of a Ca^+-dependent adenosine triphosphatase (ATPase) in the erythrocyte membrane was first reported by Dunham and Glynn (1961) who A noted that the simultaneous presence of Mg^+ and a Ca^+ increases the total ADP hydrolysis by isolated membranes from human erythrocytes, and at the same time inhibits the Na+, K+ ATPase. This observation was confirmed by Hoffman (1962), who concluded that the site of Ca^+ inhibition of the Na+, K+ ATPase UNIVERSITY OF IBADAN LIBRARY 29 - TABLE 2 Calcium transporting systems in biological membrane Transport System Membrane types References ATPase Plasma membranes Schatzmann 1966 Sarcoplasmic Reticulum Hasselbach and Endoplasmic Reticulum M1a96k1i.nose, Exchangers Plasma membranes N a +/ C a 2+ exchanger SReeiuttze,r a1n9d 6 8. except mature erythrocytes• Inner mitochondria Carafoli, Tiozzo membrane Lugli, Grovetti (Na+/Ca2+;H+/Ca2+) and Kratzing, 1974. Channels Plasma membranes Fatt and Ginsborg 1958. Electrophorectic I Vasington and Uniporters me nmnberr anMeistochondria Murphy, 1961. UNIVERSITY OF IBADAN LIBRARY - 30 - is on the inner side of the membrane. However, it was five years later that Schatzmann (1966) showed that the ATPase actually transported 2CaT* out of erythrocytes. The different kinds of plasma membrane of eukaryotic cells containing the calcium pump are shown in Table 3* It is now widely recognized that the activity of the plasma membrane Ca2 — pump is of critical importance to the maintenance of cellular Ca* homeostasis (Schatzmann 1982; Carafoli, 1981), this pump also catalyses the ATP dependent exchange internal Ca2^+ for external H+ (Niggli, Sigel and Carafoli, 1982; Smallwood, Waisman, Lafreniere and Rasmussen, 1983), and is responsible for the maintenance of a 3,000 - 10,000 fold Ca + concentration gradient across the plasma membrane. Previous, attempts to study the properties of the enzymes in unfractionated erythrocyte ghosts were difficult because of the presence of a Mg +-ATPase in the membrane. Solubilization and purification of the ATPase had been difficult due to the lability of the enzyme (Niggli, Ronner, Carafoli and Penniston 1979) its low concentration (Graf, and Fenniston, 1977) UNIVERSITY OF IBADAN LIBRARY 31 - TABLE 3 Calcium pumps in the plasma membrane of eukaryotic cells Cell type Approximate molecular Reference ___________ mass______________________ Skeletal muscle Michalak, Famulski and Carafoli 1984; Mickelson, Sarcolemma 140,000 Beaudry and Louis 1985; Sulakhe, Drummond and Ng 1973. T-tubules nd Brandt, Caswell, Brunschwig 1980; Hidalgo, Gonzales and Garcia 1986. Heart 140,000 Caroni and Carafoli 1980; Morcos and Drummond 1979; Caroni and Carafoli 1981; Kuwayama and Kanazawa 1982; Lamers and Stinis 1981; Tuana, Dzurba, Panagia, Dhalla 1981. Smooth muscle 140,000 Morel, Wibo and Godfraind 1981; Popescu and Ignat, 1983; Wuytack, Deschutter and Casteels 1980; Wuytack et al 1981. Kidney tubules 140,000 Desmedt, Paris, Borghgraefan< Wuyfack 1981; 1983; 1984; Ghijsen, Gmaj and Murer 1984; Gmaj, Murer and Carafoli 1982; Gmaj, Murer and Kinne 1979; Gmaj, Zurini, Murer and Carafoli 1983; Moore, Fitzpatrick, Chen and Landon 1974. Nervous cells Squid axon ND Dipolo 1979. UNIVERSITY OF IBADAN LIBRARY 32 - Cell type Approximatemolecular Reference Mass Synaptosomes 140,000 Hakim, Itano, Verma and Penniston,1982; Papazian, Rahamimoff and Goldin 1979; S1o9r8e1nsen and Mahler . Optic nerve ND Condrescu, Asses, Dipolo 1984. Neurohypophysis ND Desmedt eit al 1983. Ienptietshteilniaulm 115000- DeJonge, Ghijsen and 130,000 Van Os 1981; Ghijsen and Van Os 197S Hildmann Schmidt Murer 1982; Nellan and Popovich 1981 Vancorven, Roche and Van Os 1985; Vaisman Walters and Weiser./ 1988. EPnadnoccrreianse ND Kotagal, Patker landt; McDonald and Colea 1982* Pershadsingh, McDaniel, Lamdt, Bry, Lacy and McDonald 1980. Exocrine 100,000 Ansah, Molla and Katz Pancreas 1984 . Bayerdoerfer, Streb, BcKhardt^ Hasse and Schulz ,1984 Imamura and Schulz 1985. Ad ipocytes ND Pershadsingh and McDonald 1979; 1980. Osteoblasts ND Shen, Kohler and Peck 1983. UNIVERSITY OF IBADAN LIBRARY 33 Cell type Approximate Reference MMaoslsecular Leucocytes Lymphocytes 150,000 Lichtman, Segel Licht- man 1981^ SGaarrkdaodsi ,1 98E0n.yedi and Monocyctes ND Morimoto, Birge, Shen and Avioli , 1985J Scully, Segal and Lichtmann 1982. Neutrophils ND Ochs and Reed 1985; Prentiki Wollhem, and Lew.l984j Volpi, Naccache and Sha'afi 1985 Macrophages 152,500 Lew and Stossel I980j Schneider, Mottola and Romeo 1979• Ehrlich ascites ND Klaven, Pershadsingh cells Henius, Laris, Long and McDonald 1985; Spitzer, Bohmer and Grosse 1985. Plant cells ND Dieter and Marme 1981; Gross and Marme 1978; Marme and Dieter 1985: Nguyen and Siegenthaler 1985. UNIVERSITY OF IBADAN LIBRARY 34 Cell type ApproximateMolecular Reference Mass Liver 70,000-105*000 Bach, Famulski, Mirahelli and Carafoli 1985; Chan and Junger 1983? Iwasa, Iwasa and Krishnaraj. 1983! Iwasa, Iwasa, Higashi Matsui and Miyamoto 1982; Kraus- ' Friedmann Biber, Murer and Carafoli 1982; Lotersztajn, Hanoune, and Pecker 1981. UNIVERSITY OF IBADAN LIBRARY 35 1981) and the fact that the ATPase has a molecular weight and solubilization properties similar to those of Band 3» the anion channel, one of the most abundant components of the erythrocyte membrane (Ronner, 1978). Purification of the enzyme was carried out by applying affinity chromatographic techniques which involved the coupling of calmodulin to a sepharose 4B matrix (Niggli, Penniston and Carafoli 1979; Gietzen, Tejeka and Wolf, 1980). The CaM column was loaded with a Triton X-100 solubilizate of erythrocyte ghosts from which endogenous CaM had been removed by EDTA washing. The detergent, column and elution buffers contained phosphatidyl choline (PC) to maintain the Ca^+ ATPase in the bilayer state. Elution of the column was done with EDTA buffer, the EDTA -elution peak was shown by SDS-polyacrylamide gel electrophoresis to have a major polypeptide of Mr 138,000 dalton. The properties of the Ca^+ ATPase of erythrocyte plasma membrane (Table 4) resemble, in many respects, the analogous enzymes in the sarcoplasmic reticulum UNIVERSITY OF IBADAN LIBRARY 36 - TABLE 4 Properties of the plasma membrane Ca2+ -ATPase Property References Mr (SDS—PAGE) 130,000-140,000 VNiilglglail obeto eaTlT, al1,9 79 Wang et. .al, 1988 1986 Papp, et aJL, 1989. Calculated molecular weight 129,500-139.000 Shull and Greeb 1988; Verma et. al., 1988 K0.5 (Ca) -low4 -2a0f fjiinMity mode: WNainggg lie t eta,l ,a l.,1 9819981: Villalobo et ai, 1986; Wang et, al, 1989. -hig0h. 2-a0f.f7i njiutMy mode: Papp et ai, 1989; Enyedi et ai, 1987; Kosk-Kosicka and Bzdega, 1988. (ATP) -low affinity site: Wang et .al, 1989 120-290 juM -high affinity site: Villalobo et ai, 1986. 0.9-4 juM Ca2t : H+ ratio i:l , Villalobo and (electrogenic) Roufogalis 1986; KRoumwearyoa maan, d 19O88;_r_ti» z 1988* Ca2+: ATP ratio: 1:1 Carafoli 1991; Wang (reconstituted et al. 1989; Niggli et al. 1981; Romero and Ortiz 1988. k0.5 (Calmodulin) 2-6nM Niggli ejt ai, 1981; Villalobo et, ai, 1986; Wang et, ai, 1988; Kosk-Kosicka and Bzdega 1988. UNIVERSITY OF IBADAN LIBRARYino.* - 37 - of muscle cells except that it is regulated by calmodulin. The plasma membrane Ca^+-pump is the largest of all known P-type ion motive ATPases i.e. those that forms an acyl phosphate intermediate during the reaction cycle (Knauf, Proverbio and Hoffmann, 197*0 • The calculated molecular weight is between l29-139KDa (Strehler Streuler-page, Vogel and Carafoli 1989). Specific activities reported for the purified pCart- ATPase preparations range from 9.0 - 186 ;umoles mg prot hour , compared to the usual values of 0.3-3.0 ̂ lmoles mg prot -1 hour —1 for whole membranes. (Gietzen £t. al. 1980, Niggli, Adunyah and Carafoli 1981). The purified enzyme preparation when reconstituted into artificial liposomes is activated by calmodulin about 7 fold, in analogy with the findings on the membrane bound enzyme, also for the reconstituted enzyme the Ca^+ : ATP ratio is 1:1 (Carafoli 1991; Wang, Roufogalis and Villalobo 1989). Calmodulin shifts the purified and membrane bound ATPases from a low Ca^+-affinity state (Km for Ca^+ about h-20 juM) to a high Ca2+- p affinity state, (Km for Ca + about 0.2-0.7 idl) (Niggli, Adunyah, Penniston and Carafoli, 1981; Wang, 1989; UNIVERSITY OF IBADAN LIBRARY 38 - Villalobo, Brown and Roufogalis 1986; Wang, Villalobo, Roufogalis, 1988; Enyedi, Flura, Sarkadi, Gardos and Carafoli, 1987). Also the Km for the ATP is shifted from a low ATP-affinity state (Km for ATP 120-290uM) (Wang £t al, 1989; Villalobo et_ al 1986), to a high ATP state (Km for ATP 0.9 - 4 jaM) (Villalobo and Roufogalis, 1986, Kuwayama 1988; Romero and Ortiz 1988; Clark and Carafoli, 1983). 1:3 Primary structure of the Ca^-translocating ATPase The elucidation of the primary structure of plasma membrane Ca2 + pumping ATPase posed a unique problem to researchers despite the fact that the enzyme had been obtained in extremely pure form for many years (Niggli et al, 1981). These initial difficulties arose because of 2 main reasons. Firstly, the enzyme occurs in extremely low concen­ tration in the membrane and secondly, it is larger than all other P-type ATPases known. Attempts to determine the primary structure of the protein UNIVERSITY OF IBADAN LIBRARY 39 therefore focused on the sequencing of fragments of important domains of the protein. It was indeed in 1987 that, Filoteo, Gorski and Penniston successfully isolated and sequenced a tryptic fragment containing the fluorescein isothiocyanate (FITC) binding domain. This domain is assumed to be part of the binding site for ATP. To obtain the tryptic fragment, these workers labelled the purified human erythrocyte Ca^-pump with FITC and then cleaved the enzyme with trypsin. The fragment containing the label was separated by High Performance liquid chromatography. The domain had the sequence F-S-K-G-A-S-E of which K-G-A residues are highly conserved in all the P-type ion-motive ATPases. Almost simultaneous with the sequencing of ATP binding site was the determination of the phospho­ rylation site. The sequencing of the phosphorylation site was based on the fact that the phosphorylated intermediate of majority of P-type ATPases is an aspartyl phosphate located in an highly conserved sequence stretch of amino acids. Thus James, Zvaritch, UNIVERSITY OF IBADAN LIBRARY 40 - Hakhparamov and Carafoli (1987) isolated and sequenced a Cyanogen Bromide (CNBr) digest peptide corresponding to the phosphorylation domain. This sequence contains the predicted aspartic acid as the phosphate group acceptor C-S-D-K-T-G-T and it is flanked on both sides by the amino acid sequence corresponding to all other ATPases of the same group. The sequencing of the calmodulin binding domain has been much more difficult. The use of a bifunctional photoactivable cross linker has made the sequencing possible,(Fig.l) the Denny-Jaffe reagent was linked through an oxysuccinimide moiety at one of its ends to a lysine residue of calmodulin following linking. Calcium stimulated CaM was covalently bound to its binding site in the pump through photoactivation of arylazide located at other end of the reagent. The reagent was then cleaved with dithionite at the azo linkage located between photoactivable and oxysuccinimide. CaM was removed by dialysis, while the pump was still radioactively labeled. The labeled pump UNIVERSITY OF IBADAN LIBRARY 41 Pig.1 THE DENNY-JAFFE REAGENT Denny and Blobel 1984 UNIVERSITY OF IBADAN LIBRARY 42 was cleaved by Cyanogen Bromide (CNBr) and the fragment was separated by HPLC. The labeled fragment contained the sequence NH2 —E—L—R—R—G—Q—I—L-W—F—R—G—L—N—R- I-Q-T-Q-I-K-V-V-N-A-F-S-S-L-H-E-F. Shortly afterwards, the determination of the complete primary structure (Fig.2) became possible. Shull and Greeb (1988) made use of oligodeoxynucleotides corresponding to a highly conserved amino acid sequence of the ATP binding site of known P-type ATPases as probes to screen a rat brain cDNA library while Verma, Filoteo, Standford, Wieben, Penniston,Strehler, Fisher Heim, Vogel , Mathews Strehler-page, James, Vorherr, Krebs and Carafoli (1988) used oligodeoxynucleotides, but this is designed on the basis of the amino acid sequence of 2 short specific peptide from human erythrocyte Plasma Membrane Ca^+ ATPase (PMCA) as probes to isolate the corresponding cDNA. The translate< sequence contain 1220 amino acids with calculated of molecular weight^l34,683 daltons (Verma et al.1988). The location of the functional domains of the pump is now established from its complete cDNA UNIVERSITY OF IBADA LIBRARY 1 M G D M A N N S V A Y S G V K N S L K E A H D G D F G I T L A E L R A L M E L 41 R s T D A T R K I Q E s Y G D V Y G I C T K L K T 3 p N E G L S G N p A D L E R 81 R E A V F K K N F I P p K K P K T iL L JL L V W E A L D V T L I I L E i A A i v| 121 S TUG L S P J l y Q P p E G D N A L c G E V S v G E E £ G 2 G E T G |W I E G A a i 161 L s VNV C V V L V T A F N D w s K E K Q P /1—3 L Q s R I E Q E Q V F T V I R G G 201 Q V I Q I P V A D I T V G D I A Q V K Y G D L L P A D G I L i Q G N D L K I D E 244 S s L T GIVE s D H V K K S L D K D p L L L S G T H V R E G S G R M V V T A V G V281 N s Q T G I EI F T L L G A G G E E E E K K D E K K K E K K N K K Q D G A I E N R321 1 ] K A K A Q D G A A M E M Q P Tj K S E ER G G D G D E K D K K K A N L P K K E K S 361 V L Q G K L T K L A V Q I G K A G L L M S !* i T V I i L V L 1 F V i D T F W V Q 401 K R P w| L A X_JT C T SP I Y I Q Y F V| K P P I T G V T V L V V A V p E G L P L A| V T441 i S L A Y S V K K M M K D N N L V R H L D A C E T M G N A T A i C S K T G T L 481 T ®M N R M T V V Q A YI I N E K H Y K K V P E P E A I P P N I L s Y L V T G I S V 521 N V A Y T S K I L P P TE K E G G L P R H V G N K T E C A L L G L L L D L K R D Y 561 Q D V R N E I P E E A L Y K VOY T F N S V R K S M S T V L K N S D G S Y R I F S601(K) G A S E i I L K K C F K I L S A N G E A K V F R P R D R D D I V K T V I E P M641 A S E G L R T I C L A F R D F PFA G E P E P E W D N E N D I V T G L T C I A V V681 G I E D P V R P E V P D A I K KG>C Q R A G I T V R M V T G D N I N T A R A T A T 721 K C G I L H P G E D F L C L E G K DIFBN R R I R N E K G E I E Q E R T D E T w P761 K L R V L A R S S P rp D K H T L V K G I I D S T V s D Q R Q V V A V T G D G T N 801 D G P A L K K A D V 4F A M G i A G T DAV DA K E A s D T I L T D D N F T S I V K841 A V M W G R N V Y D s I S K k L Q p Q L T V NAV V A V i V A F T G A c I T Qi D S881 P L K A ]V Q M L W V N L I M D T L A s L A L A T E P P T E S L L L R K P Y G R N921 K P L I S R T M M K N I L G H A p Y Q L V V V F T L L P A G E K P D I D S G R 961 N A P L H A P P S E |H Y T I V P N T F V L M 0 LNF N E I N a | r K I H G E R N V F 1001 E G I w N N L I F C T I V L G T F V V Q I I T V Q p G G K p p S C S T? L S l E 1041 !iL L„ w..-SL_I_ T, C- M G _T_ T L Q■Li w__Q_o_ jj JL S_jrj i p T S R L K p i_- K E C7 H G T 0 E rP |Ezr E 1081 i p E E E L A E D V E E I D H A E R eIIl. R R G I0 I L w FBR G L N R I 0 T 0 i R V 1121 V N A F R S S L Y e| g L E K p E s R s s I H N F M T H ? ERF R I E‘D S E P H I P 1161 L I D D T D A E D D A P T K R N s © p p P S P N K N N N A V -D S G i H L T I E M 1201 N K S' A T S S S P G S P L H S L E T s L 1220 AR putative L~ a2 + D, i.naing aomams putative tYrans-membrane helices I I calmodulin binding domain O FITC binding site O cAMP dependent phosphorylation site O phosphorylation site Fig. 2: The complete primary structure of plasma membrane C*2+-ATPase- 44 primary structure (Fig. 2). There are three important functional domains: the sequence around Asp-4-75 where the phosphorylated intermediate is formed (James at al, 198?) and the sequence around lysine 601 where the ATP antagonist fluorescein* isothiocyanate (FITC) is bound « (Filoteo, et al 1987). The pump also contains near its C terminus, the domain where calmodulin is bound. This domain corresponds to amino acid residues 1100-1127 (James, Kalda, Fisher, Verma, Krebs, Penniston, Carafoli, 1988). This domain however, shares the same structural features, compared to other calmodulin binding peptides. These include the predominance of basic residues (Arg-1101, -1102, -1109, -1113, -1119, -1125) the presence of an aromatic residue (Trp 1107) the predominance of hydrophobic residues in the N-terminal portion of the domain, the presence of a serine-threonine cluster in the second half of the domain, and the ability to form an amphilic oC-helix. UNIVERSITY OF IBADAN LIBRARY 45 Further studies have indicated, that the calmodulin binding domain (Fig. 3) is flanked by two regions bearing a strong negative charge (residues 1079-1094- and 1153-1170). In the case of the 1079-1094- residues which was identified as domain A, this is found upstream of the calmodulin domain, it was speculated that because of the position and the charge of this stretch it was believed that it may interact with the calmodulin-binding domain (domain C) and that removal of the domain C by interaction with calmodulin (or by proteolysis) frees this domain to bind C&* with the expected high affinity. The other strongly acidic regions which is part of domain B is downstream of domain C containing residues 1153-1170. However, it is not certain, whether domain B contributes to a high affinity Ca^+ because any cleavage that removes the calmodulin domain would also remove domain B and the enzyme is known to still retains high Ca^+ affinity even affer the removal of domain C (Zurini, Krebs, Penniston and Csrafoli;l984-; Benaim, zurini and UNIVERSITY OF IBADAN LIBRARY 46 Fig.3: Proposed model for the overall topology of PMCAs. A planar representation of the PMCA is shown, including the putative transmem­ brane topology (TM 1 to 10) and the assign­ ment of important domains. Open rods and black cigar-shaped bars correspond to putative alpha-helices, and arrows denote beta-sheet s(eN co9n0dKaDr/y8 5KsDtr/u8c1tKuDr.aNl 7e6KlDe)m enatnsd. C-Ttheer mNi-ntaelrminal location (C 90KD, C 85KD C 81KD) of the tryptic cleavage sites leading to the produc­ tion of major proteolytic fragments is also indicated, as are the sites of calpain attack i(nC altphle -p rCeasMe)n ceo f (cCaallmplo du+l iCnaM,) Thaen d siatbes enocfe satetcaocnkd ariys, lcaabellmeodd ul2indn -Cianldpe(p.e+nCdaeMn)t., ACc,a lpaaciind ic Cr,eCg-itonesr mifnluasn;kCianMg, tchea lcmaoldmuoldiunl-ibni ndbiinngd indgo madiomnain; consisting of subdomains A and B; N,N-terminus: T.transduction domain;P(S), region containing the serine residue susceptible to phosphoryla­ tion by the cAMP-dependent protein kinase; PL. phospholipid-sensitive region. Adapted from Carafoli 1990. UNIVERSITY OF IBADAN LIBRARY N90kD/85kD/81kD portions of the molecule. The sequence also UNIVERSITY OF IBADAN LIBRARY - 47 - Carafoli, 1984). Several studies have shown that demains A and B, have sequence similarities to calmodulin but they have no EF hand structure (Table 5). Further downstream from the C-terminal end from domain C, there is phosphorylation site for the cAMP-dependent protein kinase (-Arg-Asn-Ser- Ser-1178). Recently, phosphorylation of serine- 1175 by this kinase has now been confirmed by protein sequencing (James, Pruschy, Vorherr, Penniston, and Carafoli, 1989). This portion of the molecule contains other regions with a high proportion of Thr and Ser residues and short sequences which fullfill the requirements for acceptor sites for the cAMP-dependent protein kinase. Conclusively, the domain may be thought of as a "regulatory region" with regulation occurring by a number of mechanisms such as calmodulin binding or cAMP directed phosphorylation. The region of Glu 296 and Lys-375 seems to account for greater spacing between the hydrophobic regions of the N-terminal portions of the molecule. The sequence also UNIVERSITY OF IBADAN LIBRARY 48 - TABLE 5 Ca^* pump sequences showing similarities to mammalian calmodulin 1079 EEIPEEELAEDVEEIDHAERE 1099 Domain A 2 DQLTEEQIAEFKEAFSLFDKD 22 Calmodulin 1141 IHNFMTHPEFRIEDSEPHIPL1DDTDAEDD 1170. Domain B 105 LRHVKTNIGEKLTDEEVDEMIREADIDGDG 154 Calmodulin 15 GVKNSLKEANHDGDFGITLAELRALM 58 Domain E 84 EIREAFRVFDKDGNGTISAAELRHVM 109 Calmodulin Carafoli, 1990 UNIVERSITY OF IBA AN LIBRARY 49 - contains two stretches which are very rich in Glu and Lys residues: the first contains a consecutive sequence of 14 residues (residues 296-309) made up entirely by Lys, Glu and an Asp. The second contain, (residues 310-321) from the same region, and this is similar to the loop of Ca^ binding regions of calmodulin, troponin C and parvalbumin i.e. an EF hand type sequence C \ (Kretsniger 1980). Residues 22-23, which is found near the N-terminus of the sequence, is also known to resemble the EF hand sequence, is also known to resemble the EF hand sequence. These regions are called domain D (296-321) and domain E (residues 22-33). Unlike domain A, B, E which have similari­ ties to calmodulin, domain D is not similar to them (Table 5) which shows the sequences of the Ca 2+ pump that are similar to calmodulin. But, it is shown that domain D and E are similar to the EF hand sequence of calmodulin. Based on the earlier speculation by Verma ejt al (1988), that domain A binds to domain C, and that domain C, when it binds CaM became free and this can now bind Ca^+ with the expected high affinity. Enyedi, Vorherr, James UNIVERSITY OF IBADAN LIBRARY 50 McCormik, Filoteo, Carafoli, Fenniston, 1989) now confirmed that the calmodulin binding domain of the plasma membrane Ca^+ pump interacts both with calmodulin and with another part of the pump. These workers also indicated that the C-terminus of the domain C strengthens the domain C-pump interaction perhaps by binding to another part of the molecule. It is speculated that the other part of the molecule might be the domain A, based on the reasons stated earlier, Vorherr, James, Krebs, Enyedi, McCormick, Fenniston and Carafoli (1990) further confirmed that indeed, the domain C binds to domain A and thus limiting its accessibility to bind to calcium (Fig. 4a). They based their work on the suggestion made by Klee (1980) that work done on a number of calmodulin regulated enzymes (Benaim §£ al, 1984; Fearson, Wenttenhall, Means, Hartshore and Kemp, 1988) has shown that the calmodulin binding domain, or a portion of it (Benaim et al, 1984), may function as a natural inhibitor for the enzymes limiting access UNIVERSITY OF IBADAN LIBRARY 51 - Fig. 4a: Hypothetical scheme of the function of the calmodulin binding domain in the plasma membrane Ca^+~pump. Adapted from Vorherr .et al, 1991* UNIVERSITY OF IBADAN LIBRARY 52 - to the active site. However in the case of the plasma membrane CaB cnA « Site C ; - options: full inclusion, partial D inclusion, deletion A cDNA examples: N Site A: rPMCA2(b):42 bp deletion Site C : hPMCAIa: 154 bp inclusion „ Site D: rPM CA l a: 33 bp inclusion hPMCA2(f):42 bp inclusion hPMCAlb: 154 bp deletion rPM CAlL e: 3 hPMCAIc: 87 bp inclusion I3 bp deletion hPMCAId: 114 bp inclusion B Site B: hPMCA4b: 108 bp inclusion hPMCA4g: 108 bp deletion R• A Fig. 6: Domain assignment in the plasma membrane calcium pump R and location of isoform variable regions. Y - 66 - 1988). Finally, Strehler ejt _al (1989) have suggested that inclusion of 87 nucleotides or 114nt of the 154 bp exon gives rise respectively to PMCAlc (1249 amino acids) and PMCAld (1258 amino acids residues), these have both been detected in human skeletal muscle. Further studies have shown that, these mRNAs from a single gene (hPMCAlb) however could encode Ca^ pump isoforms that differ in their C-terminal regulatory domains. More recently, a new Ca^+ pump isoforms were generated by alternative splicing of rPMCA2mRNA by Adamo and Penniston (1991). The inclusion or exclusion of 229 base pairs at the same position as the alternative splicing at site C is operative in both genes. These workers further observed that different isoforms which are known as rPMCA2w, rPMCA2y, rPMCA2x and rPMCA2z originate on splicing at site A. It is therefore suggested that the splicing causes the insertion of 0, 14, 31 or 45 amino acid residues near the lipid binding domain of the mole­ cule. However, the distribution of rPMCA2mRNA UNIVERSITY OF IBADAN LIBRARY 67 varies among the tissues. It has been shown that in brain and heart the rPMCA2z isoform predominates; rPMCA2w is found in kidney and uterus while brain is the only tissue which expresses important amounts or rPMCA2x. These 229 bp inclusions occur in the middle of the calmodulin domain, thus changing the length of this domain and affecting calmodulin and its binding site. Up to date no other isoform has been found in the case of the Pig smooth muscle, which also has 1220 amino acids. (De-Jaegere, Wuytack, Eggermont Verboomen and Casteels, 1990). A clear distinction and similarity therefore emerges between rat brain and human teratoma sequences Firstly is the similarity in the catalytic domains (the aspartylphosphate site and secondly is the ATP (FITC) binding site). The so called "hinge" domain (Brandi, Green, Korczak and MacLennan, 1986) that connects the two previous sites permiting them to come close in space during the functional cycle, is also similar and well conserved. UNIVERSITY OF IBADAN LIBRARY 68 - The isoforms also share the same essential topological features i.e. in both species 80% of the total weight of the pump is located outside the membrane domain, protruding into the intracellular space. The major difference appears in C-terminal region, mostly occuring in the second half of the calmodulin-binding domain (Shull and Greeb 1988). The N-terminal half on the other hand is well conserved and shows absolutely no variation among isoforms and across species (Table 7)« Its sequence is LEEGQILWFBGLNEIQTQ (Table 7 ). It has been shown that calpain can cleave at ...NE... to produce a 125 KDa fragment that is still capable of binding and stimulated by calmodulin (Wang et al 1988); James et al, 1989). This however suggested that the N-terminal half of the calmodulin-binding domain is very important. But at the C-terminal half of the calmodulin-binding sequence, there is a degree of variation only Val and phe are highly conserved out of the 9 residues. UNIVERSITY OF IBADAN LIBRARY 69 - TABLE 7 Calmodulin-binding domain of various isoforms of the Ca^+-ATPase enframe N-termCialmodulint (Subdomnaailn Ah)a lf SeqCu-etnecremsi nal half References(Subdomain B) rPMCA 1 LRRGQILWFRGLNRIQTQ IRVVNAFRSS Schull and Greels 1988. hPMCA 1 LRRGQILWFRGINRIQTQ IRVVNAFRSS Verma et al 1988. rPMCA 1a LRRGQILWFRGINRIQTQ MDVVNAFQST Shull and Greels, 1988. hPMCA 1a LRRGQILWFRGINRIQTQ MDVVNAFQSG Verma et al 1988} StreHler et al, 1989. rPMCA 2 LRRGQILWFRGINRIQTQ IRWKAFRSS Greeb and Shull 1989. rPMCA 3 LRRGQILWFRGINRIQTQ IRVVKAFRSS Greeb and Shull, 1989. rPMCA 3a LRRGQILWFRGINRIQTQ IRWKAFRSS Greeb and Shull, 1989. hPMCA 4 LRRGQILWFRGINRIQTQ IKVVKAFHSS Strehler et al 1990. bPMCA 4a LRRGQILWFRGINRIQTQ IDVINTFQTQ Mann, Brandt, Sisken and Vanaman 1990. pPMCA 1 LRRGQILWFRGINRIQTQ IRVVNAFRSS DeJaegere et al, 1990. PMCA stands for plasma membrane Ca^+-ATFase. Species origin is denoted by the preceding letter (r-rat, h-human, b-bovine - pig), the number that follows stands for isoforms e.g. rPMCA 1 is human isoform 1; alternative splicing is followed by "a" e.g. rFMCA 1a. UNIVERSITY OF IBADAN LIBRARY 70 It seems from the foregoing that, highly conserved sequences are likely to represent domains essential for the basic catalytic and transport function and may also reflect specific constraints, imposed upon structural elements of the enzyme. In contrast, however, the highly divergent sequences probably specify isoform-specific regulatory and functional specializations of the pump that are adapted to the physiological needs of the tissue/ cell type in which the corresponding enzyme is expressed. 1 :6 Mechanism of the Ca^* ATFase pump The catalytic mechanisms of all P-type ion motive pumps, has been shown to begin with the Ca^+ dependent transfer of the terminal phosphate of ATP to the pump, with the formation of a phosphory- lated intermediate (Katz and Blostein 1975; Knauf et al 197*0. The mechanisms of the Ca^+ ATPase is similar to that of all other P-type phosphorylated intermediate formed during its catalytic cycle. (Pederson and Carafoli 1987a, b). It has been shown that the Ca^+ dependent phosphorylation of the UNIVERSITY OF IBADAN LIBRARY - 71 - membrane bound pump is very rapid (Rega and Garrahan 1975; Garrahan and Rega, 1978; Schatzmann and Burgin, 1978) thus allowing the differentiation of the Ca^-ATPase from other phosphorylatable proteins of the erythrocyte membrane. Several workers have established the principla characteri­ stics of the subsequent transport cycle which can be summarized in the scheme preseted in Fig. 7 (Richards, Rega and Garrahan, 1978; Szasz, Hasitz, Sarkadi and Gardos 1978; Burgin and Schatzmann, 1979; Muallem and Karlish, 1979; 1980; Rega and G1a9r8r8ahan, 1986 and Adamo, Rega and Garrahan,). Rega and Garrahan (1986) showed that E^ is the only conf.ormer of the enzyme that catalyzes rapid phosphorylation from ATP. Several group of workers, such as Krebs, Vasak, Scarpa and Carafoli (1987) in the experiment on the mechanisms of this enzyme, showed that the membrane-bound enzyme undergoes E-)- E2 conformational transitions during the catalytic cycle and that the equilibrum between these conformational forms is influenced by various UNIVERSITY OF IBADAN LIBRARY 72 - Ca ATP V Ca E-|ATP CaE-|P !\ A •2 tt CaE2P J Ca2++ Pi Fig. 7. Scheme for (Cc?-fMc )̂ ATPase transport cycle UNIVERSITY OF IBADAN LIBRARY 73 ligands which preferentially react with the enzyme in either of the two states. In connection to this, Adamo et al (1988) observed that on pre­ incubation of red cell membrane with either Ca^+ plus Kg + or calmodulin induces a large increase in the initial rate of phosphorylation and hence, postulated that low concentration of E^ is induced by the absence of these ligands but their presence increase its concentration promoting the conversion p of E2 to E^. The role of Kg + in the catalytic cycle is its involvement in the conversion from to Exj rather than acceleration of the Ca^+-dependent phosphorylation as previously suggested (Garrahan and Rega, 1978; Adamo £t al, 1988). Furthermore the E^ form of the membrane-bound and purified enzyme p is shown to be stabilized by Ca^+ ions (Krebs et al, 1987; Wrzosek, Famulski, Lehosky and Pikila, 1989) p while in the absence of Ca^+, orthovanadate (Krebs gt al 1987) or EGTA (Vrzosek et al, 1989) shifts the conformational equilibrum to and stabilizes the E2 form. In addition, Wrzosek and his group in UNIVERSITY OF IBADAN LIBRARY 74 - the same year, were able to used circular dichroism and steady-state fluorescence methods to demonstrate that the E/j-Eg transition of the purified enzyme involves a relatively major refolding of the polypeptide chain. These workers, concluded from their data that the change in conformation of the enzyme differ because of the interaction of this enzyme with calmodulin or phosphatidyl serine (PS) Based on the hypothesis that only E^ catalyses phosphorylation and that in the absence of ligands, near 90% of the Ca +-ATPase is in the Eg conformation, Adamo e_t al (1990) estimated the relative abundance of the two conformers of the enzyme and the rates of their interconversion by measuring the initial velocity of phosphorylation of the enzyme at 37°C. These workers, suggested that in intact membranes, Cao^ -stabilized E^ possibly through Cao^+-transport sites and that the Ca2 +-induced Eg-E^ transition was strongly accelerated by Mg + . The reaction cycle starts all over, again. UNIVERSITY OF IBADAN LIBRARY - 75 - 1 :7 Modes of activation of plasma membrane Ca^*-pump Work during the last several years has shown that the plasma membrane Ca^-pump, which is one of the targets of calmodulin activation (Gopinath and Vinzenci, 1977) can also he activated by a number of alternative treatments (Table 8). These include the effect of acidic phospholipids and polyunsaturated fatty acids (Niggli et al, 1981), controlled proteolytic treatment with a number of proteases Zurfai e£ al, 1984; Benaim et̂ al, 1984), intracellular Ca^* dependent protease calpain (Vang e_t al, 1988; James et̂ al 1989) and phosphory­ lation by the cAMP-dependent protein kinase (Caroni and Carafoli, 1981; Neyses, Reinlib, and Carafoli, 1985)* Protein kinase C has also been shown to activate the pump (Smallwood, Gugi and Rasmussen, 1988). More recently it has been shown that the pump can,, be activated by oligomerization (Kosk-Kosicka, Bzdega and Johnson, 1990). UNIVERSITY OF IBADAN LIBRARY 76 - TABLE 8 Effects of various modulators on the plasma membrane Ga p+-ATPase Activator Effects References Calmodulin Increase Vmax decrease K(Ca) VGionpciennath zi a(n1d9 77); 0 .5 to 0.4-0.5 Jarrett and *lM NPiengngilsit oent; al(,1 97179)8 1; VillaloFo eT al 1986 Acidic Increase Vma* Niggli et al, 1981; phospholipid decrease Fapp _et al~^989 Ko.jjCCa) t0 Enyedi et_ al 1987 O.^liM become insentive to CaM. Protein Increase Vmax Caroni and Carafoli kinase A decrease 1981; Dixon and Haynes; K0.5(Ca) to 1989; James et al 1989. 2nM remain sensitive to CaM. Calpain Increase Vmax Wang et al 1989; 1988 decrease Au 1987; James et al Ko.5(Ca) to 1989. 0.4-0.7juM become insen­ sitive to CaM. UNIVERSITY OF IBADAN LIBRARY Activator Effects References Protein kinase C Increase Vmax Smallwood et al Ko.5(Ca) 1988. unchanged remain sensi­ tive CaM. Oligomeri­ zation Increase Vmax Kosk-Kosicka, decrease K0.5 Bzdega and Johnson (Ca) become insensi tive 1990; Vorherr, to CaM. Kessler, Hofmann and Carafoli 1991* UNIVERSITY OF IBADAN LIBRARY - 78 - 1 :8 Modulation of Ca^-ATPase by calmodulin The activation of the PMCA dates back to 1973 when Scharff and Schatzmann (1973) demonstrated that the specific activity was depressed in erythrocyte membrane isolated from EDTA concen­ tration buffers in compared to membranes isolated in the presence of calcium. Based on these findings (Scharff and Foder 1977) proposed that the Ca^+-pump exists in 2 conformations A and B. The former is believed to be induced by the presence of chelators in the medium. The state of conformation A is characterized by low Ca^+ affinity and transport rate. State B is thought to be induced by the presence of micromolar (pM) concentration with a high affinity and transport rate. It became obvious that the transition from A to B is as a result of the removal of an activator (Farrance and Vincenzi, 1977* Hanahan, Taverna, Flynn and Ekolm, 1978* Scharff and Foder,1978) when it was discovered that the erythrocyte cytosol contained a protein activator of the pump (Bond and Clough, 1972). Jarret and Penniston (1977) and UNIVERSITY OF IBADAN LIBRARY - 79 - Gopinath and Vincenzi (1977) demonstrated that it is calmodulin. The protein is a single polypeptide with a molecular weight of 16,700, has 148 amino acids, thermal stable and acidic in nature. Some 30 percent of its amino acids consist of aspartate and glutamate, this accounts for the ionic strength (PI) of 4.3. The protein also contains no cysteine, hydroxyproline or tryptophan, but it does contain a trimethylated lysine at position 1 1 5. A tertiary structure highly flexible to interact with its receptor proteins has been attributed to lack of cystine and hydroxy proline/calmodulin displays a distinctive ultra violet absorption pattern with five peaks at 253, 259, 365» 269 and 277 nm and a shoulder at 282 nm probably because of the high ratio of phenylalaline (eight residues) to thyrosine (two residues). The protein has now been isolated and purified from other varieties of sources including invertebrate and vertebrate and animal species as well as from both lower and higher plants. (Table 9). In addition, the chain of calmodulin has 4 domains (No. 1-1V) each binding UNIVERSITY OF IBADAN LIBRARY 80 - TABLE 9 Representative organisms from which calmodulin has been isolated. Organisms Classification References A) Plants Angiospermae Watterson, Sharief spinach and Vanaman 1980 Peanuts Angiospermae Anderson Charbo- nneau, Jones, McCann and Cormier 1980. Chiamydomon as Chlorophyta Gitelman and Witman 1980. B) Lower Animals Amoeba Protozoa Kuznicki, Kuznicki and Drabikowski, 1979. Englena Protozoa Kuznicki et al 1979. Tetrahymena Protozoa Jamienson et al 1979. Sea anemone Coelenterata Tazawa Sakuma and Yagl 1980. Renilla reni- Coelenterata formis (Sea Jones, Besch, pansy) Fleming, McConna ughey and Watanabe 1979. Sea Urchin Echinodermata Garbers, Hansbrough, Radany, Hyne .and Kopf 1980. UNIVERSITY OF IBADAN LIBRARY - 81 - Organisms Classification References Higher Animals Cow Mammalia Vang and Desai e1t9 76a;l , Vatterson 1980. Human Mammalia Jarrett and Penniston 1978. Pig Mammalia Yazawa et al, 1980. Rabbit Mammalia Nairin and Perry 1979. Rat Mammalia Dedman, Jakson Schreiber and Means 1978. Sheep Mammalia Autric, Perraz, Kilhoffer, and Demaltte 1980. UNIVERSITY OF IBADAN LIBRARY 82 p p one Ca^+ ion and the four Ca^+-binding sites have a dissociation constant ranging from 4 to 18 )iM. (Lin, Liu and Cheung, 197*0• Each binding site is a loop containing aspartate and glutamate side chains that form ionic bonds with Ca^+. Also, the oxygen atoms on the side chains of threonine, serine, thyrosine and asparagine residues also participate in Ca^+ binding. The structure of calmodulin is called oC -helix E Ca — binding-loop-S<-helix F where the Ca^+ binding loop is the EF hand. Fig. 8 shows the sequence of bovine brain calmodulin. Calmodulin itself is not active, its active p form is the calmodulin Ca —. complex (Lin et al 197*0 • Consequently, once it is bound to Ca<;+, calmodulin assumes a more helical conformation to become the active species, which binds reversibly to the apoenzyme resulting in the formation of an active holoenzyme. UNIVERSITY OF IBADAN LIBRARY 83 Calmodulin + Ca 2+ ■ ■ (Calmodulin * . Ca*P"+) Ê ^less active + (Calmodulin*. Ca^+)active — ..— : (E) (Calmodulin Ca^+)active (Lin et al, 1974)• Over the years, calmodulin has been found to be involved in many cellular functions including enzyme regulation, regulation of cell cycle and calcium transport across plasma membranes (Table 10.) v However, the general interpretation therefore is that the effect of calmodulin on the rate of the pump is the increase of its turnover* Calmodulin has also been found to stimulate both the rate of phosphorylation of the pump and that of its dephosphorylation (Jeffery, Roufogalis and Katz 1981, Luthra, Watts, Scherer and Kim 1980; Muallem and Karlish, 1980). UNIVERSITY OF IBADAN LIBRARY 84 - TABLE 10 Calmodulin mediated enzymes and processes Enzyme/Process References Cyclic nucleotide phosphodiesterase Cheung, 1970. Adenylate cyclase Brostrom et al,l977» Myosin light-chain kinase Wolff, Cook, Goldhammer, and Berkowctz 1980; Walsh, Cavadore, Vallet and Denaille 1980; Nairn and Perry, 1979. (Ca^++ Mg^+)-ATPase Jarrett and Penniston, 1978. Ca^+-transport Larsen and Vincenzi 1979. Phosphorylase kinase Cohen, Cohen, Shenolikai Nairn and Victor, 1979* NAD+ Kinase Anderson et al, 1980. Phospholipase A2 Wong and Cheung, 1979. Microtubule assembly Welsh Dedman, Brinkley and disassembly and Means, 1978, 1979. Neurotransmitter release Grab, Berzins, Cohen, and Siekevitz, 1979 Cell cycle regulation Chafouleaa, - Wade, Hiroyos, Aubrey and Antheny 1982. UNIVERSITY OF IBADAN LIBRARY 85 Farther studies have shown, that the tryptic cleavage of calmodulin in the presence of Ca^* results in two main fragments which have been identified by analysis of the amino acid composition as 1-77 and 78-14-8 (Guerini, Krebs, and Carafoli, 1984) while, only fragments 78-148 and 1-106 are still able to stimulate the purified Ca^+-ATPase of erythrocytes. This fragment cannot stimulate the calmodulin-dependent cyclic nucleotide | phosphodiesterase (Newton, Oldewurtel, Krinks, Shiloacl and , Klee, 1984). This study shows however the importance of the carboxyl-terminal half of calmodulin and especially of Ca^+-binding region III (Fig.8) in the interaction of calmodulin with the Ca — ATPase and provides clear evidence that calmodulin interacts differently with different targets (Guerini et al 1984). In the presence of p p Ca^*, calmodulin stimulate the C a~ ATPase activity of human red cell membranes, as well as active C a ^ transport by membrane vesicles (Bond and Clough 1973; Gopinath and Vincenzi 1977; Larsen and Vincenzi 1979). Similarly, it appears that, one calmodulin UNIVERSITY OF IBADAN LIBRARY o UNIVERSITY OF IBADAN LIBRARY 87 - 6 binds per ATPase polypeptide (Graf and Penniston 1981), although this appears to be the case in most studies (Hinds and Andreasen 1981), a 2:1 calmodulin-to-pump binding ratio has also been found in studies using the purified erythrocyte enzyme (Zurini, et al, 1984)# . lhe stimulation seems to result from an increase in both Ca^+ affinity and the maximal turn over rate of the enzyme (Scharff and Poder, 1978). However, with the use of calmodulin-depleted erythrocyte membrane preparations, the apparent Km (Ca^+) of the pump has been found to decrease from values in excess of 30 jiM to below 1 MM; the maximal rate of transport may increase up to 10 times (Jeffery, et. al 1981; Larsen, Katz and Roufogalis 1981; Larsen and Vincenzi 1979; Muallem and Karlish, 1981). Q --. . 1 :9 Activation of Ca — pump by lipids Evidence that Ca^*-.ATPase requires phospho­ lipids for activity was obtained from studies on delipidated membrane C a ^ p u m p (Roelof sen and UNIVERSITY OF IBADAN LIBRARY 83 - Schatzmann, 1977; Ronner, Gazzotti and Carafoli, 1977i Niggli at al 1981). It was shown that calmodulin is not unique as an activator of the enzyme. Like in calmodulin stimulation the trans- ition from a low to the high Ca^+-affinity state can he induced, by a variety of acidic phospholipids (Phosphatidyl serine, phosphatidyl inositol and phosphatidic acid). These phospholipids increase the Vmax 8131(1 decrease the Km for Ca^+ of the isolated enzyme to the same extent as calmodulin. Furthermore it was shown that, the activation by acidic phospholipids was due mainly to an increase of the Ca2+ sensitivity of the pump with smaller effects on the turn over of the enzyme Niggli et al 1981; Aljobore and Roufogalis, 1981) since a similar increase in Ca2+ sensitivity is seen in the activa­ tion by calmodulin. It was suggested therefore that the activators are amphiphilic in nature, since both contain acidic groups and a hydrophobic component (Gietzen, Sadorf and Bader, 1982). Also, the activation by acidic phospholipid is kinetically indistinguishable from the activation by calmodulin UNIVERSITY OF IBADAN LIBRARY 89 (Niggli ejfc al_ 1981; AlJobore and Roufogalis 1981) and the molar ratio of phospholipid to Ca^+ transport ATPase is considerably higher than that for an equivalent activation by calmodulin (Niggli et al 1981). Based on these observations it was suggested that the mode of binding of these two activators are not identical. Further experiments showed that, the stimula- tion of the Ca^l_ATPase activity by negatively charged phospholipids is based on a binding of these lipids to the Ca^i-ATPase and that the negative charges are a major modulatory factor for this interaction (Verbist,Theodurus, Gadella, Racymaekers, Vuytack, Virtz and Casteels, 1991). However, it has been shown that the actual position of the lipid regulatory domain may be tentatively placed at the N^-terminal 5KDa part of the 81 KDa tryptic fragments (Papp, at al 19897 as suggested by the change in the transport kinetics, and also by the large number of positively charged residues in this area in the amino acid sequence of the plasma membrane calcium pump (Verma £t al, 1988). Kore UNIVERSITY OF IBADAN LIBRARY 90 - recent work has shown that, the site of phospho­ lipids interaction is at the C-terminal portion of the highly charged stretch of 44 amino acid residues separating the N-terminus of the fragment of 76 KDa from those of the 81, 85 and 90 KDas (Zvaritch eb al_ 1990). Further studies have shown that, there is another domain for phospholipid interaction apart from the one stated by Zvaritch et al 1990)j the other domain, is at the calmodulin binding domain, (Brodin e_t al_, 1991). On the other hand, there are some lipids that inhibit the enzyme, such as oleic acid which was shown to competitively inhibit calmodulin activation of red cell ghost enzyme and compete with calmodulin binding (Wetzker, Klinger and Frunder, 1985) although this does not preclude competition at an allosteric site or at overlapping site. UNIVERSITY OF IBADAN LIBRARY - 91 - 1:10 Activation of the purified Ca^-ATPase of the erythrocyte membrane by controlled proteolysis The stimulation of the membrane bound calcium pump by proteolysis was first shown by Taverna and Hanahan (1980) in which they used chymotrypsin and trypsin on isolated erythrocyte membranes. However, a detailed study of the phenomenon was performed shortly thereafter on inverted erythrocyte vesicles by Sarkadi, Enyedi and Gardos (1980). They were also able to show that trypsin mimicked the effects of calmodulin. It was observed that the activated pump was no longer stimulated by calmodulin. Further studies have indicated that the treatment reduced the steady state level of the phosphorylation in the absence of Mg2+. Stieger and Schatzmann (1981) have shown that the activation by trypsin corresponds to a decrease of the Km (Ca +) of the pump to a level that is much lower than that of calmodulin. Moreover, when it UNIVERSITY OF IBADAN LIBRARY - 92 - is maximally activated, the pump became fragmented into a number of products, the most prominent having molecular weights of ^ 100, KDa and 4-0 KDa. Fragments in 1981 Stieger and Schatzmann suggested that>the calmodulin receptor was the fragment of 40 KDa that was removed from the main body of the pump, because there was a loss of calmodulin sensitivity and this confirmed tha work done by Enyedi, Sarkadi, Szasz,Bot and Gardos (1980). Zurini,Krebs, Penniston and Garafoli (1984), did a comprehensive study on the controlled tryptic fragmentation of the purified erythrocyte enzyme and this has led to a rapid fragmentation of approximate molecular weights of 90 KDa, 81 KDa and 76 KDa. •fragments it was shown that 1 ^ ^ lodoazido calmodulin orosslinked to the 90 KDa fragment product, thus showing that calmodulin interacting domain was contained in a fragment of 9 KDa fragment removed from the 90 KDa product as proteolysis reduced it to 81 KDa fragment. This is in contrary to the study of (Stieger and Schatzmann 1981) which indicated that about 40 KDa fragment is removed. It also showed that UNIVERSITY OF IBADAN LIBRARY - 93 - the treatment almost immediately removed from the enzyme a fragment of molecular weight 33 KDa which interacted better than any other of the cleavage products with the hydrophobic probe M- 125 lodophenyl diazirine. This fragment was suggested to contain hydrophobic intramembrane regions of the pump. The acylphsphate inter­ mediate was not formed by products having molecular weights lower than 76 KDa fragment. A product of 45 KDa fragment which most likely was derived from the 76 KDa fragment bound a radioactive ATP analogue and was thus suggested to contain the ATP binding site. Benaim, Zurini and Carafoli (198*0 were able to add significant information to the organisation of the pump, this is as a result of the milder conditions they used. They observed that an additional 65 KDa fragment and activitymeasurements revealed it to be less responsive to calmodulin than the product of molecular weight 90 KDa fragment (Benaim, Clark and Carafoli, 1986; Benaim et al, 1984). However, thse two different proteolysis conditions seemed to promote the and E2 conformations. The UNIVERSITY OF IBADAN LIBRARY - 94 - prbeolysis scheme is shown in Fig. 9. Further­ more, and based on the work ef Benaim al (1986), it was possible to deduce that the products 90 KDa and 89 KDa fragments bound calmodulin,wwhereas the product of 81 KDa fragment did not bind calmodulin. It was also shown that the product of 76 KDa had higher activity in the absence of calmodulin. The 90 KDa fragment responded normally to calmodulin the 89 KDa fragment was less able to do so, despite the fact that it could still bind it. It seems therefore probable that the calmodulin binding domain acts as an inhibitory sequence i.e. it would bind to a domain in the pump that is essential for full action as earlier explained. Zvaritch, et jal (1990) were able to give a conclusive support for the trypsin proteolysis scheme in (Fig. 9). Trypsin proteolysis has also been used to map the functional domains in the plasma membrane Ca^+-pump (Fig. 10). This showed that the 90 KDa fragment produced the C-terminal sequence ASK, and it corresponds to residue 1161 in the structure of human plasma membrane calcium ATPase 4 (hPMCA 4) isoform and UNIVERSITY OF IBADAN LIBRARY 95 - CaM binding subdomain * Trypsin in the presence of CaM 85 KDa J CaM m + CaM N N- K Trypsin in the presence of VO_3“- + Mg2+ 81 KDa \ + PL M S M l 76 KDa Trypsin in EGTA (EDTA)I MU Fig. 9s Scheme for trypsin proteolysis of purified Ca 2+- pump in the presence of different effectors. Adapted from Carafoli, 1991. UNIVERSITY OF IBADAN LIBRARY 96 - 90-85-81 KDa 35-33.5 KDa (N-terminus). (C-term in us) 1 16 19 308 \ Acety|- M [ ^ ] ESREGD E N R N K ^Q D G V A LE i 314 315 Q 33.5 KDa (N-terminus) PL 67 76fK-D-a- -(N---te-rm--inNus) GAKGIQVALRTLKGQLVSKEKKPVKVAKKDKEENDIGEQ 359 rD4f75 f(P)x K609 (ATP) 76-31 KDa (C-terminus) AGHGTTKEEITKDAEGLDEIDHAE M c 1066 E, 85 KDa (C-terminus) K'Q NYPKQISEi 1105 90 KDa (C-terminus) H S FMTHPEFAIEEELPRTPLLDEEEEENPDKAsJ: F G 1161 T R 1205 ______1171 v VSTEL - - { j |GDLLLV calmodulin binding domain | putative transmembrane domains Fig. 10: Location of NH2 and COOH termini of tryptic fragments of molecular masses 90, 81 and 76 KDa. Adapted from Carafoli, 1991. UNIVERSITY OF IBADAN LIBRARY 97 - the cut occurs Just outside the calmodulin binding domain (Verma et al, 1988; Strehler James, Fischer, Heim, Vorherr, Filoteo Penniston and Carafoli 1990). While the 85 KDa fragment produced the C-terminal triplet K1Q and the cut is at residue 1105 and within the calmodulin binding domain. Whereas the 81 and 76 EDa fragments gave the same TTK C-terminal sequence with certain amounts of it in the background, the cut in this case is at residue 1067 and it completely removes the calmodulin binding domain which means the difference in the properties of the two fragments resulted from the opposite end of the fragments. It could be suggested that the attack by trypsin, in producing the 76 EDa fragment (44 amino acids downstream of the N-terminus of the 81-EDa fragment) structurally modifies, that portion of the pump by preventing it from interacting with phospholipids "In addition, it could confer to it a state which makes the pump optimally responsive to Ca^+ in the absence of phospholipids. Subsequently, the K-terminal sequence of the functionally inactive, UNIVERSITY OF IBADAN LIBRARY - 98 - tryptic fragments of 35 and 33«5 KDa were also determined (Zvaritch et_ al^ 1990). However, in the case of the 90 KDa fragment the cut was at position 315 while the 83 and 81 KDa fragments coincided with that of the 90 KDa fragment and the region is rich in basic residues, their cuts wereeat positions 358 and 34-8. But in the case of the 78 KDa fragment this occurred further downstream in the human isoform of the pump (Zvaritch et al, 1990) and the cut is at leucine 359 while the cut 35 KDa and 35.5 KDa fragments were at residue 19. It is not known, whether trypsin attack between putative transmembrane helices 2 and 3 i.e., the fragments of 33.5 - 35 KDa are removed from the main body of the pump in the absence of SDS. However, it was suggested that the properties of the products resulting from cleavages between transmembrane helices 2 and 3 reflect those of the entire sequence of the enzyme from the N-terminus to the cleavage point(s) in the hydro­ philic domain protruding from the 10th putative transmembrane helix. UNIVERSITY OF IBADAN LIBRARY - 99 - 1.11 Modulation of Ca^-ATPase by calpain It is well established that, calcium dependent neutral cysteine proteases, collectively called calpain, regulate cellular functions by limited proteolysis of specific proteins. For instance the protease is involved in the degradation of myofibrillar protein (Dayton, Goll, Zeece, Robson and Reville 1973), activation of protein kinase C (Melloni, Pontremoli, Michetti, Sacco, Sparatore and Horeckes 1986), limited proteolysis of erythrocyte membrane cytoskeleton proteins (Lang, Wickenden, Wunne and Lucy), degradation of B-hemoglobin in erythrocytes (Pontremoli, Melloni, Sparatore, Michetti and Horecker 1984) and the modification of calmodulin-binding proteins Wang, Villalobo and Roufogalis (1989). This protease is present as an inactive proenzyme (procalpain) which can be converted through Ca +-dependent autoproteolysis to a mature form (Calpain fully active at micromolar Ca^+-concentration. UNIVERSITY OF IBADAN LIBRARY 100 - (Pontremoli and Melloni, 1986). The procalpain is composed of a catalytic 80 KDa subunit and a 30KDa subunit (Pontremoli and Melloni, 1986). It was shown that, the primary structures of both human subunits have been deduced from the nucleotide sequences of cDNA (Suzuki, 1987) and found to contain, distinct domains. (Fig. 11) shows the domain structure of calpain. In the case of the 80KDa it is composed of four domains (I, II, III, IV) whereas the 30KDa subunit has two (V and IV). Domains IV and i v ' are homologue and have four consecutive EF hands (Kretsinger, 1975)* Two different forms of calpain have been demonstrated. The characterization of these forms was based on their Ca + sensitivities (Suzuki 1987; Pontremoli, Melloni, Murachi 198^. Calpain 1 and Calpain 2 are active only at micromolar and milliomolar. Independently Au (198^) and Wang at al (1988), found that treatment of the plasma membrane-bound Ca^+-ATPase with calpain 1 activated the Ca^+-ATPase activity in an irreversible UNIVERSITY OF IBADAN LIBRARY -101 - Catalytic $0 K DA subunit dcoymsteaiinne protease CCaal-mboindduilnin -like100 T 300 50Q g, do main 700 I cys-1i0i 8 His-265 III IV Regulatory 30KDA s ubunit Gly-rich Calmodulin-like Ca-binding hydrophobic domain domain Fig .11. Domain structure of Calpain Suzuki, 1987 UNIVERSITY OF IBADAN LIBRARY 102 - manner and rendered it calmodulin insensitive. However, in the case of the calpain activation, the Km for Ca^+ is shifted from a value of 6 juM for the native ATPase to 0.5 UM for calpain digested species. Assays are performed in the presence of calmodulin and no appreciable difference is found between the native and calpain digested Ca^+-ATPase, the Km for Ca^+ being O.J/iM for both enzyme. Further studies have shown that there is consistent structural modification with respect to the native enzyme species, either purified or membrane- associated, but in the absence of calmodulin, the basic change is the conversion of the native l38KDa Ca^+-ATPase to a 124-KDa fragment devoid of the calmodulin-binding domain while a 127KDa fragment retaining its calmodulin-binding domain is produced in the presence of calmodulin (Wang at alL, 1988; James at al, 1989). Although, calpain cleaves and leaves a fragment of 124KDa in the absence of calmodu­ lin, studies, have also shown that, calpain actually UNIVERSITY OF IBADAN LIBRARY 103 - cuts at 2 different portions of the Ca^+ ATPase molecule. The first cut occurs in the middle of the calmodulin binding domain producing a fragment of about 14KDa and a (calmodulin binding) fragment of about 124 KDa while a second cut occured closer to the N terminus of the calmodulin binding domain producing a fragment of about 124 KDa and accounted for the loss of calmodulin binding at prolonged times of incubation of the ATPase with calpain. The 124 KDa fragment has been demonstrated to have an increased rate of Ca^+ transport in a liposome reconstituted system (Wang et al, 1989). 1:12 Activation of Ca^* pump by kinase mediated phosphorylation Smallwood and Rasmussen (1988) have shown that, the activated form of purified protein kinase C was found to stimulate Ca^+ transport in alkaline phosphatase-pretreated inside out vesicles (I0VS) suggesting that the Ca^+ ATPase may already be partly phosphorylated in the cells in Situ. This UNIVERSITY OF IBADAN LIBRARY 104 - activation is however in the presence of specific activators such as phorbol ester and diacylglycerol and the physiological concentrations of free Car* was between 0,08 - 5uM. Some other studies have indicated that both, protein kinase C and phorbol ester increased the maximum velocity of erythrocyte I0V Ca^+ transport but had no significant effect A upon the apparent Km for Ca^+. In contrast, calmodulin increased both the maximum velocity and the Ca^+ sensitivity of the pump while a combina­ tion of the two activators (protein kinase C and 12-0-tetradecan©yl phorbol-13-acetate (TPA) or protein kinase C and diolein a phospholipid ̂ * plus calmodulin showed additive effects at maximal doses of each and suggesting therefore that calmodu­ lin and protein kinase C may exert their effects through different mechanisms (Smallwood and Rasmussen 1988). In addition, these workers, have shown that protein kinase C and TPA or protein kinase C and diolein have been found to stimulate the activity of the erythrocyte purified calcium pump ATPase. (Fig. 12) shows a proposed model of the activation UNIVERSITY OF IBADAN LIBRARY 105 - I 2H0H Pig. 12: Model of the erythrocyte membrane calcium pump. Smallwood et ad, 1988. UNIVERSITY OF IBADAN LIBRARY 106 - of Ca2^-pump by protein kinase C. According to this model, in an intact cell and in its basal state * Calmodulin may bind to and activate the Ca^+-pump, almodulin further stimulates the pump when intracellular Ca^+ is elevated, elevation of cytosolic Ca^+, in combination with the production of diacylglycerol thus enables endogenous protein kinase C to bind to the plasma membrane and this in 2+t urn, phosphoryr lates the pump. Excess cytosolicCa may then be extruded. It is suggested that, although Ca^+ concentra- tion will fall toward$^basal level both calmodulin and FKC would enable the pump to remain in an activated state. It is predicted that the Ca^+-ATPase would be dephosphorylated by an endogenous phosphatase as yet uncharacterized when influx of Ca^+ ceases and itdacylglycerol is no longer generated. Further work has been done, to characterize the particular site at which the protein kinase C phosphorylates. In this regard Wang, Wright, Machan, Allen, Conigrave and Roufogalis (1991) demonstrated that the phosphorylation site is located within a UNIVERSITY OF IBADAN LIBRARY - 107 - 12KDa a region at the carbonyl terminal end of the molecule and that the single threonine within the calmodulin-binding domain is a likely site of phosphorylation. Another serine residue located at the carboxyl-terminal to the calmodulin binding domain has been suggested to be a possible additional phosphorylation site. In the same work, a mixture of isozymes (£>(, p, Y ) o f FKC was shown to activate the purified Ca +-ATPase by 5-15%» this activation was increased from 40 to 60% when A23187 was included in the assay on the other hand the -isozyme of PKC alone appears not to activate but to reduce activation by calmodulin (Wang et_ al, 1991). It is suggested that the differences in activation might have various explanation firstly, FKC could phospho- rylate the purified Ca^-ATFase only substoichiome- trically thereby producing only a moderate activation. Secondly, PKC could also phosphorylate directly or indirectly other membrane components, possibly including some phospholipids, which could as well indirectly stimulate the Ca +-ATPase and, thirdly it seems likely that, the solubilized and purified Ca^+-ATPase UNIVERSITY OF IBADAN LIBRARY 108 - may not allow the full expression of the effect of PK-C phosphorylation. Furthermore, the plasma membrane Ca 2+-ATPase from both heart sarcolemma and erythrocyte cells are also activated as a result of the phosphoryla­ tion by cAMP-dependent protein kinase A in vitro (Caroni and Carafoli 1981; Neyes e_t al, 1985). Recent work has demonstrated that PK-A phosphorylation leads to increased affinity for Ca 2+ as well as a 2 fold increase in Vmax (Dixon and Haynes 1989; James, Pruschy, Vorherr, Penniston and Carafoli, 1989) (Table 8). It is worth: noting therefore, that in the erythrocyte the increase in Vmax produced by PK-A is non-additive to that of calmodulin (James et al, 1989). In contrast to protein kinase C, cAMP-dependent protein kinase was shown to phosphorylates the plasma membrane Ca 2+-ATPase at a single serine residue located between the calmo­ dulin-binding domain and the carboxyl terminus of the enzyme about 5 KDa from the carboxyl terminal end (James et al, 1989). It was therefore speculated that activation could be achieved in like manner by UNIVERSITY OF IBADAN LIBRARY 109 - modulation of calmodulin-binding domain. 1:13 Activation by Oligomerisation Another way by which the Ca^+-ATPase is stimulated is through dimerization or oligomeri­ zation (Kosk-Kosicka, at al 1989)* It has been shown thatea dimerized pump could form the trans- membrane ion channel between two subunits, whereas a monomeric pump would have to possess an intra­ molecular pathway for the translocations of the p enzyme. Using the sarcoplasmic reticulum ATPase as a model, Hymel, Maurer, Berenski, Jung and Fleisher (1984-) suggested that Ca^+ pump could exist as a dimer. Thiswas later confirmed by Anderson (A1989). Its seems plausible, that sarco- plasmic CeT* ATPase could exists as a dimer because of its low molecular weight and high concentration in the membrane. However, in the case of the plasma A membrane Qâ "‘-ATPase this is not likely, because of its high molecular weight and low concentration of the enzyme (0.1%) in the membrane (Knauf, et al 197*0 • Therefore it is possible that, the occurence of the dimer would be something more specific rather UNIVERSITY OF IBADAN LIBRARY 110 than a randomized collision-mediated association process. Further studies by Kosk-Kosicka et_ al_ (1989) using fluorescence resonance energy transfer showed that energy transfer occurred between enzyme molecules, the effect being half maximal at 10-20juJN. It was suggested that concentration dependent stimulation is due to oligomerisation. These workers demonstrated further that 1 mole of calmodulin binds to 1 mole of monomeric Ca^+-ATFase whereas only 0.5 mole of calmodulin was bound to 1 mole of the oligomeric form of the enzyme. These findings suggest that the notion that at high concentration, the enzyme most likely exist as dimers. Further studies, by Vorherr, Kessler, Hofmann and Carafoli (1991) reveal that plasma membrane Ca2^+-ATPase truncated at the COOH terminus by calpain to a fragment of 124 KDa does not contain the calmodulin binding domain could not oligomerize with that intact ATFase, suggesting UNIVERSITY OF IBADAN LIBRARY Ill - that calmodulin binding domain mediate the process of oligomerisation of the Ca^+-pump. 1 .11+ Historical Background Intensive research on the aflatoxins started in I960 when the mysterious Turkey 'X' disease killed young turkeys, ducklings and young phesants in England and certain parts of Africa (Blount, 1961). About the same time an outbreak of trout hepatoma was reported in the U.S.A. (Wolf and Jackson, 1963). Earlier on, Burnside, Sippel, Forgacs, Caril, Atwood and Doll (1957) had reported a fatal disease characterized by liver lesions in swine fed on mouldy corn. Sargeant, Sheridan and O'Kelly (1961) and Spensley (1963) eventually showed that the new disease was a mycotoxicosis caused by the ingestion of groundnut meal infested with the moud Aspergillus flavus. Almost simultaneously Hartley, Nesbitt and O'Kelley (1963) showed that this mould when cultured in appropriate medina produced toxic metabolites collectively called "aflatoxins or toxins of Aspergillus flavus. UNIVERSITY OF IBADAN LIBRARY - 114 - 113 - TABLE 11 Local (Nigerian) Foodstuffs known to support aflatoxin production. Medium Aflatoxin metabolites produced by Aspergillus flavus strains Soybeans »1 Bg G1 G2 Paw Paw B1 B2 G1 G2 Beans (Black eye) B1 B2 G1 G2 Banana B1 b2 - - Groundnuts B1 B2 G1 G2 Red pepper B1 B2 - - Millet B1 b2 G1 G2 Rice B1 b2 G1 g2 Corn B1 b2 G1 g2 Tams (Yam flour) B1 b2 G1 g2 Garri (Manihot flour) B1 b2 G1 g2 UNIVERSITY OF IBADAN LIBRARY 114 - chromatopiates were designated aflatoxins B^, B2 , , and G2 in order of decreasing Rf value? because only relatively small amounts of the toxins were available, the structural elucidation made use of modern organic chemistry relying almost solely upon interpretation of UV, IF, NMR and mass spectra* Using these tools it was found that the major toxin, aflatoxin B^ exhibited a blue fluorescence, has +a melting Npoint of 268-269°C, and UV spectrum XEm^ 225, 265nm and 562nm. It's molecular weight Was found to be 512 by mass spectrometry and this correlates well with the empirical formula A comparison of the infrared spectra of aflatoxin B^, coumarin and tetrahydro-deoxoaflatoxin indicated that the two absorption bands 1760cm and 1084-cm in the spectrum of the natural product are due to the presence of a coumarin and ketone carboxyl groups in the molecule. Further studies using NMR technique aided the complete structural elucidation of aflatoxin B/| (Fig. 13) (Asao, Bucchi, Abdet- UNIVERSITY OF IBADAN LIBRARY t - 115 - AFLATOXIN \ Fig. 13*. Structure of aflatoxin B-j UNIVERSITY OF IBADAN LIBRARY 116 - Kader, Chang, Wick and Wogan, 1965). Reports by Vander Merwe, Fourie and Scott (1965) on the interuption of the catalytic hydrogenation of aflatoxin after the uptake of one mole of hydrogen to give a product which was identical with aflatoxin B«| confirmed that AFB2 is dihydro aflatoxin B^. The structures of other aflatoxins were easily deduced because of their similarity to that of aflatoxin B^ (Vander Merwe, 1965). These structures are shown in (Fig. 14). It has been shown that the aflatoxin G series seldom occur in the absence of B^, even though cases of the G series contaminated products have been reported (Wogan, 1977). Aflatoxins M,j and which are hydroxylation products of B-i are as toxic as the parent aflatoxin (Purchase 1967b). Dutton and Heathcote (1965) described two other hydroxy derivatives of B2 and G2 designated aflatoxin B£a and G2a. Another compound assigned the name aflatoxin Ro, was found to be less toxic than B^ (Detroy and Hesseltine, 1970). UNIVERSITY OF IBADAN LIBRARY 117 - UNIVERSITY OF IBADAN LIBRARY 118 - 16 Carcinogenesis of aflatoxin Lancaster, Jenkins and Philip (1961) were q the first group of scientistswho gave an insight * into the carcinogenic potential of the aflatoxins when they demonstrated a high incidence of liver tumours in rats fed for 30 days on diets containing a highly mouldy peanut. Extracts, from their culture media of the various mould were found to induce tumours. Subsequent studies, established that the aflatoxins were the carcinogenic agents in mouldy infected peanuts (Barnes and Butter 1964, Dickens and Jones,” 1964, Newberne >1965$ and Wogan 1966). The effective dose of aflatoxin for the induction of liver tumours in rats was estimated to be in the order of 10jjg/per day (Butler, 1965). Wogan, (1963) found that dietary aflatoxin levels equal to or higher than O.lppm would induce liver carcinoma at an incidence greater than 50%, if fed to rats everyday for up to 80 weeks. Earlier on, Wogan and Newberne (1967) had shown that a specified amount of carcinogen UNIVERSITY OF IBADAN LIBRARY 119 - given at a low dosage over a long period of time has a greater carcinogenic effect than a higher dose given over a shorter period of time. A linear correlation between liver tumour incidence and dietary aflatoxin intake over the 0.07ppm - I.Oppm have been established for the rat (Newberne, 1965). Several factors'.have been found to come into play in the carcinogenesis of aflatoxin*JOr instance; Wogan (1975) showed that female animals develop tumours more slowly than males^although the incidence of tumour is similar in both sexes. Apart from hepatomas, carcinomas in many other tissues of the rat have been reported, these lesions affect for example, the glandular portion of the stomach (Butler and Barnes 196$), and the renal tubules (Butler, Greenblatt, Lijinsky 1969). Lesions such as focal lipid accumulation, parenchyma cell hyperplasia, bile duct hyperplasia have also been reported. Furthermore, certain species of animals have been shown to be mere susceptible to aflatoxin than UNIVERSITY OF IBADAN LIBRARY 120 - others. In particular, trouts developed a 10% liver tumour incidence when fed 0.1 ug dietary aflatoxin while ducks are one of the most susceptible to the carcinogen. In contrast mice and sheep are resistant to aflatoxins carcinogenesis, despite their comparable ability to metabolize these compounds to their predominant metabolite (Bassir and Emafo, 1970; Bassir and Emerole )1975)• Histolo­ gical assessment of aflatoxin-induced tumours showed that there are two main types of tumours developed in rats fed aflatoxin, those derived from hepatic parenchymal cells which later formed nodules, and those derived from the bile duct epithelia (Lancaster, 1968). Injections of aflatoxin B^ (38%) and G>j (56%), Bg and G (traces) in arachus oil \iM2 nt oo-rats show that the animals developed sarcomas and fibrosarcomas at the injection sites (Dicken and Jones, 1964). Tumours in other animal species have suggested that the sunbird trout is more sensitive to the hepatocarcinogenic effect than the rat 0 or(Sinnhusber and Walee 1965)• In addition, Gopalan (197 described a liver carcinoma in a rhesus monkey UNIVERSITY OF IBADAN LIBRARY 121 - after feeding aflatoxin for a period of 6 months to 3 years. In man, however, the actual carcino­ genic potency of the aflatoxin has remained rather speculative although there is considerable evidence to show that several food consumed by man are contaminated by Aspergillus f1avus. 1: 17 Occurrence of aflatoxin in Some Nigerian foods So far only two species of moulds namely Aspergillus and Penicillum have been found to be very versatile in producing the aflatoxins. Mould strains obtained from the Nigerian enviroment have been shown to produce aflatoxins (Bababunmi and Bassir;1976). Previous work in this laboratory have been shown that several of the Nigerian food stuffs can serve as suitable media for the growth of these moulds (Emerole and Uwaifo, 1980), of all the food stuffs studied pawpaw gave the lowest yield of crude aflatoxin (Table This finding gave the impetus for screening some Nigerian food stuffs for possible contamination by aflatoxin. Aflatoxin screening of the chloroform UNIVERSITY OF IBADAN LIBRARY 122 - extracts of some Nigerian foods show that both the B and G series of the aflatoxins are actually present in certain food stuffs. Different levels of aflatoxin ranging from 0.04-mg in rice and 1.70mg/kg in groundnuts were found (Emerole, Uwaifo, Thabrew and Bababunmij 1982). 18 Inhibition of DNA synthesis by aflatoxin Inhibition of DNA synthesis in cells exposed to aflatoxin preparation has been demonstrated under several experimental systems, thus, under in vivo conditions, this response is particularly evident in rat liver undergoing regeneration after subtotal hepatectomy. However, Frayssinet, Lafarge, Recondo and Le Breton (196*0 reported that aflatoxin B/j inhibited the net synthesis of liver DNA when immediately after hepatectomy at dose levels of 30 or 6Qug/animal; this showed that the enzymes respon­ sible for DNA synthesis remained fully active under these conditions, hence the toxin exterted its inhibitory effects by interacting with DNA in such a way as to impact its ability to act as a primer for DNA synthesis. This was confirmed by Rogers UNIVERSITY OF IBADAN LIBRARY 123 - and Newberne (1967), who found that a single large dose (3mg/kg) of aflatoxin given to rats caused marked reduction of tymidine - 3H labeling of liver cell nuclei,* But, the evidence regarding the mechanism of inhibition is not yet conclusive, it tends to support the hypothesis that this action is a consequence of interaction of the compound with DNA. 19 Inhibition of protein synthesis by aflatoxin B^ Earlier studies have shown that the mechanisms of gene transcription and translation, alterations in DNA-dependent RNA synthesis would be expected to result in changes in protein metabolism. Therefore, it has been shown that exposure of liver tissue to aflatoxin B^ results in marked alterations in gene transcription as evidenced by impaired synthesis of nuclear RNA* Associated with this effect are significant and persistent losses of cytoplasmic RNA and polyribosoma disaggregation. Impaired synthesis of total liver proteins has been observed upon exposure of liver slices to aflatoxins in vitro. However, under in. vivo conditions synthesis UNIVERSITY OF IBADAN LIBRARY - 124 - of specific proteins (inducible enzymes) is inhibited by the toxin, whereas total liver protein synthesis is not markedly affected (Wogan, 1968) • Clifford and Rees (1963) demonstrated that the in vivo synthesis of certain liver enzymes is inhibited in animals treated with aflatoxin , characteristics of inhibition in these instances suggest that the observed effects on protein synthesis are secondary to inhibition of R M synthesis. This was however, based on studies of toxin effects on the inducibility of tryptophan pyrrolase, an enzyme present in mammalian liver at low levels (Feigelson and Greengard ,1962) ♦ Wogan (1968) however concluded that the action of aflatoxin in vivo involves suppression of the synthesis of specific liver proteins through its alteration of RNA metabolism. Aflatoxin has also been shown to cause an irreversible displacement of polysomes from rough endoplasmic microsomal, membrane (Rabin, Blyth Doherty, Freedman and Williams, 197*0 > by acting directly on polysomes and thus inhibiting mRNA UNIVERSITY OF IBADAN LIBRARY 125 - synthesis. The monosomes produced contained amino acyl transfer RNA. 1: 20 Alterations of RNA metabolism by aflatoxin Altered RNA metabolism as a result of aflatoxin B>| treatment has been demonstrated under a variety of experimental conditions. It has been shown for example that in vivo administration of the compound to rats or direct exposure of liver slice preparations in vitro results in rapid and dramatic inhibition of precursor incorporation into RNA, particularly in the nucleus (Wogan, 1968). Subsequently, the impaired RNA synthesis was attributed to inhibition of RNA polymerase activity (Gelboin, Wortham, Friedman and Wogan 1966). Further studies by Friedman and Wogan (1967) confirmed also that the toxin produced inhibition of RNA polymerase activity that persisted for several days after dosing. In view of the marked effects of the toxins on nuclear RNA synthesis, it might be anticipated that cytoplasmic RNA metabolism would be affected as well (Fr edman and Wogan 1966; Svododa and Soga 1966) UNIVERSITY OF IBADAN LIBRARY 126 - In certain studies, it was observed that the primary effect of aflatoxin is to block RNA synthesis, on binding to the DNA molecule (Hayes, Platt, Tilzer, and Chiza 1975)* Also, the intercalation of aflatoxin with the DNA template results in the release of an inhibitor which selectively inhibits the transcriptional activity of RNA polymerase II (Yu, 1977)• 21 Effect of aflatoxin on nucleolar morpholo.gy Alterations of nuclear RNA metabolism resulting from treatment with aflatoxin are associated with changes in the morphology of nucleoli as observed by electron microscopy of affected cells. However, work done by Bernhard, Frayssinet, Lafarge and Le Breton (1965) on liver cell nucleoli of rats, showed that there were some lesions. These lesions consisted of segregation of the granular and fibrillar components of the organelle, with the formation of so-called nucleolar "Cap". The structural change was developing within 30 minutes after administration of the toxin (0.5mg/kg), but proved to be reversible, since the nucleolar UNIVERSITY OF IBADAN LIBRARY 127 - morphology was essentially normal 24 hours after injection. Subsequently Lafarge, Frayssinet and Simard (1966) also confirmed the result and they concluded that inhibition of nucleolar RNA synthesis preceded development of the morphologic lesion, as the former process was essentially maximal 20 minutes after treatment with the toxin, whereas the latter took longer to develop. Wogan (1968) was able to conclude that the marked alterations in nucleolar morphology induced by single doses of aflatoxin are associated only with acute effects of the toxin but do not occur after chronic treatment. 22 Interaction with Proteins Bassir and Bababunmi (1973) demonstrated that aflatoxin B^ is firmly bound at least at one site unto either bovine or human serum albumin in vitro. It was shown that aflatoxin interacts Uhspecifically with different proteins showing the same behaviours as described for BSA (Evrain, Cittanova and Jayle 1978). Subsequently, binding of fl aflatoxin to rat plasma was investigated in vivo and in vitro. UNIVERSITY OF IBADAN LIBRARY 128 It was observed that, aflatoxin binds an apolar site with an association constant of 30mM_/1 at pH 7*4- and 20°C. The association constant was pH-dependent, increasing about 1.7 fold as the pH increased from 6.1 to 8.4. This was ascribed to a pH-induced conformational change in the albumin molecule. Thermodynamic studies,indicated that the aflatoxin-albumin interaction was exothermic ( H - -29.3 KJi mol-1), with a S value of -13.8 J. mol- 1̂ K- ”1 (Heini and Schabort, 1986). Furthermore, earlier studies have shown the effect of functional groups on the interaction of aflatoxin and with starch and cellulose; seven other cellulose derivatives were later studied by Uwaifo and Bassir (1977) using equilibrium dialysis. Their finding further emphasized the functional roles of amino groups in the interaction of aflatoxin with macromolecules. 1:23 Interactions of aflatoxins with other cellular compounds It has been suggested that aflatoxin binds to the membrane to produce degranulation of the rough UNIVERSITY OF IBADAN LIBRARY - 129 - endoplasmic reticulum Garvican and Rees (197^0 Gurtoo and Dahms, (197*0 showed that the absence of toxicity of aflatoxin B2a might be due to its binding to extra cellular proteins of the membrane, thus preventing its entry into the cell. The hydroxyl of C2 atom appears to prevent it's re-entry into the cell. It has been shown that, the OH of C2 atom does not have this same effect since the labelled forms of aflatoxin, and GN2 are incorporated into other aflatoxins. The interaction of cellular organelles has been demonstrated. For instance, the toxins interacts with some cellular organelles such as lysosomes which contain dhydrolytic enzymes such as ribonuclease (Pokrovsky, Krowchenko, and Tutlyan, 1972). Lysosomes are known to release degradative enzymes glycosidase, hyaluronidase, proteases and collagenase during tumour maliginant formation (Cameron, 1966; Balaz and Von Euler, 1952; Fiszerszarfar and Szafard 1973; Harris, Stephens, Ghosh and Taylor, 1977). Tbe persistence of hyaluronidase release in neoplastic cell proliferation is absent in normal cell proliferation. UNIVERSITY OF IBADAN LIBRARY 130 - To establish the relationships of mitochondria chemical carcinogen interactions to the normal to neoplastic transition. Graffi showed as early as (19̂ 0), that carcinogenic hydrocarbon are accumulated by the mitochondria of animal cells. In more recent years, the interactions of chemical carcino­ gens with mitochondria have been studied in vitro, and the effect of carcinogen feeding to rats on the functional properties of freshly isolated mitochondria has been studied as well (Belt and Campbell, 1973)* Aflatoxin exerts several types of effects on freshly isolated rat liver mitochondria. These include a partial inhibition of respiration, a partial inhibition of RNA synthesis, an induction of mitochondria swelling and an enhancement of uncoupler-stimulated ATPase activity (Doherty and Campbell^1973, Belt and Campbell, 1973, 1975; Bababunmi and Bassir, 1976). 1: 2k Activation and Toxicity of aflatoxin Many carcinogenic agents are electrophilic in nature and are able to react with nucleophilic UNIVERSITY OF IBADAN LIBRARY 131 - cellular constituents (Miller, 1970). It is well established that aflatoxin does not react towards nucleophiles (Sporn, Dingman, Phelps and Wogan 1966; Clifford and Rees 1967). Aflatoxin B>j 2,3-oxide was first suggested by Schoental (1970) as an intimate carcinogenic metabolite of aflatoxin B^. In general, the 2,3-epoxide acts as an alkylating « agent for macromolecules (Dickens and Jone 1964). The existence of aflatoxin 2,3-oxide/was demonstrated by Swenson, Miller, Miller (1974) on isolating the 2,3 dihydro 2-»3 dihydroxy (dihydrodiol) (Pig. 15). Aflatoxin B^ form an acid hydUrfol„lpyesLa bt Le of the r-RNA # of aflatoxin adduct form by hepatic microsomal oxidation of aflatoxin B^ in the presence of r-RNA. The instability and high reactivity of aflatoxin 2-3 oxide made it impractical to isolate it from microsomal reaction mixtures or to synthesize it chemically. Results of several studies have shown that the toxicity of aflatoxin may be classified into two; namely subacute toxicity and acute toxicity. It has been shown that adult animal would not die for several days after an intake of sublethal UNIVERSITY OF IBADAN LIBRARY 132 - rat liver in vivo rat liver AFB 1, 2,3, ox I base or nucleoside Pig. 15: The metabolic activation of aflatoxin B-j by rat liver* Swenson et al, 1975, UNIVERSITY OF IBADAN LIBRARY 133 - quantities of aflatoxins (Tulpule, Kadhavan and Gopalahan 1964-; Verret, Karliac and Maclaughlin 1964) while the acute toxicity syndrome appears after ingestion of a high dose of aflatoxin in the diet. -Symptoms of the aflatoxin toxicity include necrosis of the kidney tubules following ingestion of aflatoxin B/j or aflatoxin G^ to rat (Butler, 1964). Haemorrage lesions have been found in other organs such as the lungs and adrenal glands of rats. It has been shown that, 2 functionalities of the aflatoxin molecule are important determinants of its biological activity, as regards acute effects. Firstly substituents fused to the lactone portion of the coumarin nucleus determine activity to the extent that the aflatoxin G configuration is less potent than that of aflatoxin B. Further indication of the specific importance of this segment of the molecule is provided by the recent finding that " aflatokicol” the hydroxylated derivative produced by reduction of the carbonyl group of aflatoxin B^, is less toxic than B^ to ducklings (Detroy and Hesseltine, 1970)* Consequently, more evidence UNIVERSITY OF IBADAN LIBRARY 134 - points to the importance of the dihydrofurofuran portion of the aflatoxin molecule. Secondly, reduction of the vinyl ether double bond in the terminal furan ring brings about significant reduction in potency in most systems and nearly total loss of activity with respect to acute toxicity to rats. 1:25 Properties of aflatoxins Ultra violet light and infra-red spectra have been used for the characterisation of the physical properties of aflatoxins. The physico­ chemical properties of the aflatoxins are shown in (Table 12). The chemical properties of aflatoxins deal with the oxidation or the addition reaction they undergo. Aflatoxins are unstable in alkaline medium (Vander, Zijden and Barretj1962), in the presence of methyl lamine or ammonia (Dollear and Mann/1968) in methanol, air and ultra-violet light. Aflatoxins B^ and B2 possess a reactive carbonyl * group in their cyclopentenone ring. This takes part in ketonic reactions reacting with 2,4- dinitrophenyl- hydrazine and hydroxylamine to form phenyl hydrazones and oximes (Crisan and Grefig, 1967). Aflatoxins B,, UNIVERSITY OF IBADAN LIBRARY 135 TABLE 12 Physico-chemical properties of the aflatoxins. Chemical Molecular Fluorescence Aflatoxin formulae weight under u.v 363mu Aflatoxin B or Difurocoumarocyclopentenone series. Bl C17H12°6 312 Blue b2 C-vpSlh°6 413 Blue B2a C17H14°7 330 Blue c17h 12°7 328 Blue M2 330 Blue Ro C17H1406 314 Blue Ql C17H12°6 328 Blue pi C17H12°6 298 Blue M2a c17h 14° 8 3^6 Turquoise Aflatoxin G or Difurocoumarolactone series. G1 C17H12°7 328 Turquoise g2 °17h 14°7 7 330 Turquoise G2a C17H14°8 346 Turquoise G m C17H14°8 344 Turquoise GM2 C17H12°8 346 Turquoise &M2a C17H14°9 362 Turquoise C17H14°6 302 Blue UNIVERSITY OF IBADAN LIBRARY 136 - and G^ react with ozone (Dwarakanatk, Rayner, Mannaand Dollear, 1968), benzoyl peroxide, osmium tetroxide and potassium iodine complex (Trager and Stoloff, 1966). Aflatoxins , B2 * G^ and G2 also react with acetic acid and trifluoroacetic acid employing thionylchloride as a catalyst to form derivatives with Rf values different from that of the parent compound (Androlles and Reid, 1964; Stoloff, 1967; Crisan and Grefig, 1967). UNIVERSITY OF IBADAN LIBRARY 157 - 1 :26 OBJECTIVE The aflatoxins are highly hepatocellular carcinogenic chemicals; aflatoxin being the most potent of these toxins. Binding studies using equilibrium dialysis technique have shown that aflatoxin B^ binds firmly to serum albumin (Bassir and Bababunmi, 1975) and polysaccarides (Uwaifo and Bassir, 1977)* Further binding studies using Bacillus by>revis revealed that 66% of cell-bound // . toxin was associated with the cell membrane fraction of the bacterium (Uwaifo and Bassir, 1978). The finding that a significant amount of the toxin occurs in blood following a single intraperitoneal injection of the toxin suggests that the toxin must have contact with the membrane (Wogan and Shank, 1977)• It is not known, whether the binding of aflatoxin B«j to proteins and polysaccarides jin vitro could be extrapolated to its behaviour in the blood. It appears that if aflatoxin B>j binds firmly to red cell membrane proteins, as it does to serum albumin, the properties and functions of the membrane may be modified. Such modifications could involve transport of cations especially Câ "1" across the red cell UNIVERSITY OF IBADAN LIBRARY - 138 - membrane, since the proteins responsible for carrying these cations require membrane phospholipids in their immediate environment for maximum activity. Moreover, binding of the toxin directly to transport proteins could - significantly affect the catalytic activity of the pumps. Presently, there are 2 main types of ion motive ATPases that are present in the membrane of the red cell. The Na+/K+-ATPase pumps 3Na+ against 2K^ at the expense of ATP while Ca^+-ATPase A regulates the intracellular C & + concentration by A pumping C ar+ out of the cell (Schatzmann, 1966). A Although, Ca^+ has to fulfill several messenger functions within the cell, elevated intracellular A Ca^+ concentration has been shown to cause an increase of K+ leak transport, an inhibition of Na+, K+ ATPase, and a destruction of the plasticity and normal shape of red blood cells (Sarkadi and Tostenson, 1979). A It is now well established that Ca^-pumping ATPase is responsible for the active extrusion of Câ "*" from erythrocytes, thus maintaining the erythrocyte A intracellular concentration below 1̂ iM while the Car* level in the surrounding plasma is about 1-2mM UNIVERSITY OF IBADAN LIBRARY - 139 - (Schatzmann and Vincenzi 1969; Roufogalis, 1979). Recent studies have shown that different types of genes code for different isoforms of the pump (Shull -and Greeb 1988, Verma at al, 1988), hence they differ in their tissue specificity, localization and functions. In human, however, the most abundant is the hPMCA4 and it is the major isoform in erythrocytes. In spite of the fact that a number of substances have been shown to inhibit the calcium pump, to date no known specific inhibitor to the pump has been identified pharmacologically. From pharmacology standpoint, a specific inhibitor of the pump will be useful in controlling the intrace- llular concentration of Ca£:+. Such a drug will certainly have important value in experimental studies on the role of calcium in control of muscle contraction. Potentially, the drug may find parti­ cular use in overcoming heart failure and in increasing blood pressure in hypotension. Furthermore, availability of a specific inhibitor will alleviate the current difficulties encountered in the design of experiments aimed at evaluating the physiological and pathological roles of the pump. UNIVERSITY OF IBADAN LIBRARY 14-0 - The overall objective therefore was to study the influence of the toxin on the functional integrity of the pump and thus its effects on the regulation of intracellular Cac:+ homeostasis . . in cells using the erythrocytes as model. The effect of the toxin on the catalytic activity of the pump was studied after establishing--that the toxin binds to the red cell membrane. UNIVERSITY OF IBADAN LIBRARY 141 - CHAPTER TWO MATERIALS AND METHODS 2.1 Collection of Samples Blood samples (500ml ) were obtained from healthy individuals who visited the Blood bank of the Ibadan University College Hospital. Fresh human blood was also obtained from the Blood bank of University of Zurich Teaching Hospital, Zurich. 2.2 Preparation of erythrocyte ghost membrane Reagents (i) Isotonic Buffer: IJOmM KC1 + 10mM Tris pH 7.4 9.69g of potassium chloride, KC1 (Hopkins and Williams, England) and 1.21g of Tris (hydroxymethyl) amino methane (Sigma Chemical Co., London) were dissolved in about 900ml of distilled water and the pH was adjusted to with a E-520 model pH meter. The solution was made up to 1 litre in a standard volumetric flask and stored at 4°C. (ii) Hypotonic Buffer: 10mM Tris ♦ 1mM EDTA, pH 7»i*- * 1.21g of Tris (hydroxymethyl)-amino methane (Sigma UNIVERSITY OF IBADAN LIBRARY 142 - Chemical Co., London) and 0.380g of the tetra- sodium salt of ethylene diamine tetra acetic acid, EDTA (Sigma Chemical Co., USA) were dissolved in about 900ml of distilled water and the pH was adjusted to 7-^* The solution was made up to 1 litre and stored at 4°C. (iii) Washing Buffer; 10mM HEPES, pH 7-4- 2.38g of N-2 hydroxyethyl piperaizine-N ethanesulphonic acid, HEPES (Sigma Chemical Co., London) was dissolved in about 900ml of distilled water and the pH was adjusted to 7-4. The solution was made up to 1 litre. (iv) Storage Buffer: 10mM HEPES, 50jiM CaCl2 , 500juM MgCl2 , 130mM KC1 2.38g of N-2-hydroxyethyl piperazine-N-ethane - sulphonic acid, HEPES (Sigma Chemical Co., London). 7.4-mg of hydrated calcium chloride, CaCl2 . 2H20 or 5.5mg of anhydrous calcium chloride, CaCl2 (BDH Chemicals Ltd., England), 9«69g of potassium chloride, KC1, (BDH Chemicals Ltd., England 0.102g of Magnesium chloride hexahydrate, MgCl2. 6H20 UNIVERSITY OF IBADAN LIBRARY w - (BDH Chemicals Ltd., England) were dissolved in about 900ml of distilled water and the pH was adjusted to 7*/+* The solution was made up to 1 litre with distilled water and stored at 4°C. (v) Addition of phenylmethylsulfonylfluoride (PMSF) Phenylme thylsulphonyfluori.de (PMSF) solution in DMF was added to Buffers (ii) and (iii) at a final concentration of 0.2mM just before use. 500mM PMSF solution was normally prepared by dissolving O.^i^^g of PMSF (Sigma Chemical Co., London) in of Dimethyl formamide, DMF. Procedure Haemoglobin-free ghost membranes deficient in calmodulin were prepared by the procedure of Niggli et_ al, (1981) which is based on the principle of hypotonic lysis developed by Dodge, Mitchell and Hanahan (1965). All steps of the membrane prepara­ tion were carried out at 4°C. UNIVERSITY OF IBADAN LIBRARY Whole blood samples were centrifuged at 6.000 rpm for 10 mins in an USE refrigerated centrifuge and Damon/IEC PR-600 refrigerated centrifuge. The plasma and "huffy” layers (containing white blood cells) were removed by aspiration to obtain packed erythrocytes. The erythrocytes were washed thrice in 10 volumes of isotonic buffer pH 7*/* to remove plasma constituents. Each time, the cell suspension was centrifuged at 6.000 rpm the supernatant was always removed by aspiration. Washed erythrocytes were lysed in 10 volumes of hypotonic buffer pH 7»4, and centrifuged at 18.000 rpm for 20 minutes. Also, the washed erythrocytes were lysed in 10 volumes of hypotonic buffer 7.4 and RC 50 Sorvall Zonal centrifugation was used. Membranes were washed four times in the hemolysis solution and eight times in 10mM HEPES containing 0.2mM PMSF, pH 7»^ in order to remove calmodulin, haemoglobin and EDTA. The haemoglobin- free ghosts were finally resuspended in a storage buffer CaCl2 and stored at -80°C and used within 2 weeks. UNIVERSITY OF IBADAN LIBRARY 145 - 2.5 Determination of Protein Erythrocyte ghost membrane protein was estimated by the procedure of Lowry £t̂ a]̂ , 1951 using bovine serum albumin as standard. Principle The colour reagent used in this method is a phospho-18-molybdotungstic complex (a mixture of several molecular forms such as 5H20.P20^.9Mo03 and 3H2O.P2O5 10 WO^.SMqO^) which can be reduced by 4 phenol groups to give a blue colour at alkaline pH. Tyrosine (and tryptophan) residues present in the protein are responsible for the reduction of the phosphomolybdo tungstic complex. This complex talso called phenol reagent, is very unstable and decomposes in alkaline solutions. Since it interacts with tyrosine, only at alkaline pH, an excess of the reagent must be added in order to get complex reaction. However, these high concentrations of UNIVERSITY OF IBADAN LIBRARY 146 - the phosphomolybdo tungstic acid can cause turbidity due to the formation of insoluble salts. Folin and Ciocalteu (1927) have found that the turbidity can be prevented by adding to the reagents lithium salts (e.g. Li2 So^). In their mixture, called Folin-Ciocalteu's reagent, they also added some bromine water to maintain the phosphomolybdo tungstic reagent in the oxidized state during storage. Lowry, Rosebrough, Fan and Randall (1951) found that pretreatment of the protein sample with alkaline copper solution markedly increased the colour developed in the reduction of the phosphomolybdo tungstic reagent. Their assay system include a mixture of Na2C0^- NaOH to buffer the pH around 10 to neutralize the phosphoric acid produced by the degradation of the phosphomolybdo tungstic complex at alkaline pH. Reagents (i) Reagent A : 2% Na^CO^, 0.4% NaOH, 0.16% Na-tartrate, 0.8% SDS. UNIVERSITY OF IBADAN LIBRARY - 147 - 20g of sodium carbonate (Na2C0^) (BDH Chemicals Ltd, England), 4g of sodium hydroxide pellets, NaOH, (BDH Chemicals Ltd., England), 1.6g sodium- potassium tartrate (Na-KC^O^.^^O) (BDH Chemicals Ltd., England), 18g of sodium dodoecyl sulphate SDS (Sigma Chemical Co. London) were dissolved in a little quantity of distilled water in a 1 little quantity of distilled water in a 1 litre standard volumetric flask and made up to the mark with distilled water. (ii) Reagent B : 4% copper sulphate 1g of copper sulphate (CuSO^. JpH^O), (BDH Chemicals England) was dissolved in a small volume of distilled water and made up to the 100ml mark in a volumetric flask. (iii) Reagent C : Alkaline copper solution This was prepared fresh just before use by mixing 100ml of reagent A with 1ml of reagent B. (iv) Reagent E ; Folin-ciocalteu Reagent 100g of sodium tungstate (Na2W0^.2H20) (BDH Chemicals Ltd., England) and 25g of sodium UNIVERSITY OF IBADAN LIBRARY 148 - molybdate, (^2^100^.2^0) (BDH Chemicals Ltd., England) were dissolved in 700ml of distilled water in a round-bottomed quick-fit flask. 50ml of 85% ortho phosphoric acid (BDH Chemicals i'td., England) and 100ml of cone. HC1 BDH Chemicals Ltd., England) were added to the solution and the mixture was gently refluxed for 10 hours. To the resulting mixture were added I50g of lithium sulphate and 2 or 5 drops of Bromine water. The mixture was boiled for 15 minutes in a fume hood without condenser to remove excess bromine. After cooling, the solution was made up to 1 litre, filtered and stored at 4°C. The 2N solution of folin ciocalteu was usually diluted to 1N with distilled water just before use. Standard protein solution 10ml of 4ml/ml Bovine serum albumin (BSA) (Sigma Chemical Co., London) was prepared by dissolving 40mg of BSA in 10 mins of water. 1ml of the prepared solution was diluted with distilled water (about 16-I9ml) to obtain a solution with an absorbance of 0.140 at 279nm. UNIVERSITY OF IBADAN LIBRARY - 149 - Based on the fact that the molecular extinction coefficient (E) of BSA is 45,000 and its molecular weight is 65,000, this absorbance will give a concentration of 200 ug/ml albumin for the solution. Procedure Assay was carried out in duplicate. The reagents were dispensed into the various test tubes as outlined in the following protocol. The contents of each tube were rapidly mixed on addition of reagent C and left to stand at room temperature for 10 minutes and reagent D was later added and the contents of the tube was vigorously mixed. Mixing is very important since reagent D is unstable at alkaline pH and can be destroyed before it has reacted with the protein-copper complex. The absorbance of the solution in each tube was measured at 660nm after 45 minutes in WPA spectrophotometer. A standard curve was generated from the absorbance values obtained. UNIVERSITY OF IBADAN LIBRARY TABLE 13 ProtoQol for protein Estimation According to the Modification * Procedure of Lowry et al 1931 Test tubes in duplicates 1 2 3 4 5 6 7 8 Standard BSA solution (200jig/ml) - 100jil 200)i 1 300)il 400)il 500ja l - - Samples - - - - - - 100)11 200)il Distilled water 800jil 700jii 600)il 500jil 400)il 300)i 1 700ul 609ul Reagent C 2.4ml 2.4ml 2.4ml 2.4ml 2.4ml 2.4ml 2.4ml 2.4ml Reagent D 240)il 240jl1 240jil 240)ll 24Qul 240)il 240)i 1 240jul 150 UNIVERSITY OF IBADAN LIBRARY - 151 - 2.4 Measurement of protein concentration of purified C a A T P a s e enzyme samples Total protein concentration was determined by the procedure of Lowry e_t al (1951) as modified by Markwel, Hassi, Tolbert and Bierber (1981), using bovine serum albumin as standard of the interfering materials such as HEFES, dithiothreitol, Triton X-100 etc, the protein was first precipitated with deoxycholate and trichloroacetic acid as described by Bensadoun and Winstein (1976). Reagents 2% Sodium Deoxycholate 2g of the sodium salt of deoxycholic acid (Fluka AG, Switzerland) was dissolved in 100ml of distilled water andestored at room temperature. 24% Trichloroacetic acid 24g of trichloroacetic acid, CCl^ COOH, (BDH Chemicals Ltd., England) was dissolved in 100ml of distilled water and stored at room temperature. Procedures The protein was first precipitated using a mixture of 6% trichloroacetic acid (TCA) and 125ug/ml UNIVERSITY OF IBADAN LIBRARY - 152 - sodium deoxycholate, since TCA alone does not produce quantitative protein precipitation at low protein concentrations (1-25 ug). An aliquot of the protein sample (10-50u 1) was made up to a final volume of 3ml with distilled water. Twenty five micro litre of 2% sodium deoxycholate was tfc then added and the solution was mixed vigorously on a vortex mixer and allowed to stand for 15 mins. One mililitre of 24% trichloroacetic acid was then added and the solution was again mixed vigorously and centrifuged at 3»000Xg for 30 mins. The supernatant was gently removed by used of a pasteur pipette connected to a vacuum line; the last 0.5ml was removed gently by tilting the tube and maintaining the tip of the suction pipette in contact with the side of the test tube without disturbing the pellet. The pellet was then dissolved in a 3ml of Lowry reagent C, by mixing vigorously on vortex, and allowed to stand for 10 min. Folin-Ciocalteu reagent (D) (0.3ml) was then added, and the solution mixed rapidly and allowed to stand for 30 minutes UNIVERSITY OF IBADAN LIBRARY after which the absorbance was read at 660nm. Bovine serum albumin was used as standard. A blank containing bovine serum albumin was similarly treated. 2.5 Assay of membrane-bound ( C a H p ; )-ATPase activity Reagents (i) 9% Ascorbic Acid (Reagent A). 22.5g of L-ascorbic acid, (Sigma Chemical was dissolved in a quantity of distilled water in a 250ml standard volumetric flask and made up to the mark. The solution was then stored in a brown reagent bottle and kept in a refridgerator at 4°C. 1.25% Ammonium Molybdate in 6.5% Sulphuric acid Reagent B 6.25g Ammonium molybdate (NH^)^ 4-H2O; (Hopkins and Williams Ltd., England) was dissolved in 500ml of 6.5% sulphuric acid (BDH Chemicals Ltd., England). The latter was prepared by mixing 32.5ml of concentrated UNIVERSITY OF IBADAN LIBRARY 154 - sulphuric acid in water and making up the solution to the mark in a 500ml standard volumetric flask. The reagent was stored at room temperature in a plastic bottle. (iii) Assay Solution? 2M Potassium Chloride 14.912g potassium chloride, KC1 (BDH Chemicals Ltd., England) was dissolved in a little quantity of distilled water and then made up to the mark in an 100ml standard volumetric flask. The solution was stored in a reagent bottle at 4°C. Assay Buffer: 500pK HEPES or 500mM Tris buffer 29»7875g of N-2-hydroxethyl piperazine-N-2- ethanesulphonic acid HEPES (Sigma Chemical Co., London) was dissolved in about 200ml of distilled water and the pH adjusted to 7**» The solution was then made up to 250ml in a standard volumetric flask and stored at 4°C. In the cases where Tris yp buffer was used, 15«125g of the TrizmaQ base was dissolved in about 100ml of distilled water and 105ml of 1tt HC1 was added. The solution was adjustec to pH 7.4 and then made up to the 250ml mark in a standard volumetric flask. The solution was stored at 4°C. UNIVERSITY OF IBADAN LIBRARY - 155 - 0.1M MgClo 2.033g of magnesium chloride hexahydrate (MgCl2.6H20) (Hopkins and Williams, England) was dissolved in a little quantity of distilled water in 100ml standard volumetric flask. After this, the solution was made up to the mark with distilled water. This was quantitatively transferred into a reagent bottle and stored at 4°C. 100mH Ca012 1.4-702g of calcium chloride dihydrate (CaCl2.2H20) (BDH Chemicals Ltd., England) was dissolved in a little quantity of distilled water and the solution made up to the mark in a 100ml standard volumetric flask. This was quantitatively transferred into a reagent bottle and stored at 4-°C. 1mM CaCl2 was prepared from this stock solution by dispensing 1ml of it into water and making up the solution to the 100ml mark with water in a 100ml standard volumetric flask. The solution was also stored at 4-°C in a plastic reagent bottle. UNIVERSITY OF IBADAN LIBRARY 156 - 50mM EGTA 0.9509g of Ethylene glycol-bis-(2-aminoethyl) tetra-acetic acid (Sigma Chemical Co., Loddon) was accurately weighed and suspended in a little quantity of distilled water in a 50ml beaker. With constant stirring, potassium hydroxide solution was carefully added to it until the pH is 7«4. The solution was quantitatively transferred into a 50ml volumetric flask and made up to the mark with water. After transfer into a small reagent bottle, the solution was stored at 4°C. 10% SDS 10g of sodium dodecyl sulphate, SDS (BIO-RAD Labs. Richmond, California, USA) was dissolved in 100ml of distilled water. The solution was left to stand on the bench at room temperature. 40mM ATP 0.4842g of disodium salt of Adenosine 5 - triphosphate, ATP (Sigma Chemical Co., London)was carefully weighed and dissolved in 18ml of 10mM HEPES, pH 7.4. The pH of the solution was adjusted UNIVERSITY OF IBADAN LIBRARY 157 - to 7.4 and the solution made up to the mark in a 20ml volumetric flask. The solution was normally dispensed into eppendorf tubes (2ml aliquots) and stored at 0°C. lOrnM Na2HP04 0.14-2g of disodium hydrogen phosphate/I. (Na2HP0^)(BDH Chemicals Ltd., England) was dissolved in a little quantity of distilled water and then made to the mark in a 100ml standard volumetric flask. Calmodulin Solution 1mg calmodulin (Calbiochemicals Ltd., London) was dissolved in 5ml of 10mK HEPES pH 7 to produce a 200jig/ml solution of calmodulin. Procedure Determination of the Ca 2+-ATPase activity in the Hembrane The Ca^+-ATPase activity was assayed by measuring the rate of release of inorganic phosphate from the Y-position of ATP. The phosphate liberated to the medium was determined colorimetrically using UNIVERSITY OF IBADAN LIBRARY - 158 - the method of Raess and Vincenzi (1980) based on the procedure of Fiske and Subbarow (1925). Calmodulin-depleted membranes (l00-200jug) were incubated at 37°C for 5 minutes in a final total volume of 1ml. Final concentrations of the consti­ tuents in the assay medium was 100mM KC1, 50mM potassium-HEPES pH7.4, 2mN MgCl2, 20mM CaCl2, 4 2mM EGTA (when added), and 2jag/ml calmodulin (when added) The reaction was started by adding 2mMr- ATP (Final concentration). After 30 minutes of incubation at 37°C, with constant shaking, the reaction was stopped with 1ml of 10% solution of sodium dodecyl sulphate (SDS) in distilled water, 1ml of reagent B was then added to the reaction mixture and a blue colour was developed with the addition of 9% ascorbic acid solution. After 30 mins the absorbance of the solutions was read at 660nM. Exact timing between the addition of reagent A and reading at 660mm is critical* The activity of the Mg +-dependent ATPase (assayed in the absence of added CaCl2 i.e. in the presence of 2mM EGTA) was subtracted from the total assay in the presence of Cacr+. UNIVERSITY OF IBADAN LIBRARY 159 - 2.6 Purification of erythrocyte plasma membrane Ca^*-ATPase on a calmodulin affinity column 2M KC1 37-29g of potassium chloride, KC1 (BDH Chemicals Ltd., England) was dissolved in a little quantity of distilled water in a 250ml standard volumetric flask. The solution was made up to the mark, transferred quantitatively into a reagent bottle and stored at 4°C. 2M NaCl was prepared the same way except that 29.22g of sodium chloride (BDH Chemicals Ltd., England) was used instead of KCl. 20% Triton X-100 20g of Triton X-100 (Sigma Chemical Co., London) was dissolved in 100ml of distilled water and the solution was stored, at 4°C. lOOmM MgClo 2.034g of Magnesium chloride hexahydrate (MgCl2.6H20) (BDH Chemicals Ltd., England) was dissolved in a little quantity of distilled water in a 100ml standard volumetric flask. After UNIVERSITY OF IBADA LIBRARY 160 - dissolution, the solution was made up to the mark, transferred quantitatively into a reagent bottle and stored at 4°C. 100mft HEPES. pH 7.4 5«95g of N-2-hydroxyethyl piperazme-N - ethanesulphonic acid, HEPES (Sigma Chemical Co., London) was dissolved in / 200ml of distilled water and the pH was adjusted to 7*4-. The solution was made up to 250ml in a standard volumetric flask and stored at 4°C. lOOmM CaClp 1.471g of calcium chloride dihydrate, CaC^.S^O (BDH Chemicals Ltd., England) was dissolved in a little quantity of distilled water in a 100ml standard volumetric flask. The solution was made up to the mark, transfered quantitatively into a reagent bottle and stored at A°C. 100mM EDTA pH 7.4 18.6g of the sodium salt of ethylene diamine tetracetic acid, EDTA (Sigma Chemical Co., London) was dissolved in about 40ml of distiled water and UNIVERSITY OF IBADAN LIBRARY 161 - the pH was adjusted to The solution was made up to 50ml in a standard volumetric flask and stored at 4°C. 10mg/ml Phosphatidyl Choline lOmg of phosphatidyl choline was dispersed in a tube and was sonicated to clarity. 1ml of storage buffer (l30mM KC1, 20mM HEPES, 500jiM MgC^, 50^1 CaCl2 » pH 7*^) was later added to dissolve the sonicated phospholipid and stored at 4-°C. 500mM Dithiothreitol 7.6g of dithiothreitol (DTT) (Aldrich Chemical Co., U.S.A.) was dissolved in a about 70mls of distilled water. The solution was made up with distilled/to 100ml in a standard volumetric flask and stored at 4°C. 87% glycerol This reagent was purchased from (Fluka Chemical Co., Switzerland) and was stored at room temperature. UNIVERSITY OF IBADAN LIBRARY 162 - 0.5% Sodium Azide 500mg of sodium azide (Fluka Chemical Co., Switzerland) was dissolved in about 80mls of distilled water in a volumetric flask. The solution was made up to the mark and transfered in to a reagent bottle and stored at 4°C. A. Solubilization Buffer pH 7«^ This was prepared as follows: Stock Solution Volume used Final in Buffer Concentration preparation 1M NaCl 13ml 130mM lOOmM HEPES pH 7.4- 10ml 10mM lOOmM MgCl2 0.5ml 0.5®M lOOmM CaCl2 0.05ml O.O^iM Distilled water was added to make up to the final volume of 100ml. UNIVERSITY OF IBADAN LIBRARY 163 - B. Equilibration Buffer pH 7*4- This was also prepared as follows; Stock Solution Volume used in Buffer F Coinncaelntrat: preparation 20% Triton X-100 2.0ml 0.4% lOOmM Hepes pH 7»4 20ml 20mJS NaCl 13ml 130mM lOOmM MgCl2 1ml 1mM lOOmM CaCl2 0.1ml lOOpM 85% 11.49ml 10% 500mM DTT 0.4ml 2mM DTT $mg/ml phosphatidyl choline 50mg 500}ig/ml Distilled water was added to make up to the final volume of 100ml. UNIVERSITY OF IBADAN LIBRARY 164 - C. Washing Buffer Stock Solutions Volume used in Buffer Final preparation Concentration 20% Triton X-100 0.25ml 0.05% lOOmM HEPES pH 7.^ 20ml 20mM 1M NaCl 15ml 130mM lOOmM MgCl2 1ml 1mM lOOmM CaCl2 0.1ml 100jaM 85% glycerol 11.4-9ml 10% 500mM DTT 0.4-ml 2mM 5mg/ml phosphatidyl choline 50mg 0.5mg/ml Distilled water was added to make up to the final volume of 100ml UNIVERSITY OF IBADAN LIBRARY 165 - D. Elution Buffer pH 7.4 Stock Solutions Volume used in Buffer Final preparation Concentration 20% Triton X-100 0.25ml 0.05% lOOmM HEPES pH 7.4 20ml 20mM 1M NaCl 15ml 150mM lOOmM MgCl2 1ml 1mH 85% glycerol 11.49ml 10% 500mfl DTT 0.4 ml 2mM 5mg/ml phosphatidyl choline. 50mg 0.5mg 500mM EDTA 0.4ml 2mM Distilled water was added to make up to the final volume of 100ml UNIVERSITY OF IBADAN LIBRARY 166 - Solutions for washing calmodulin Affinity Column After Elution E. 1st washing medium Stock solution Volume use Final in Buffer Concentration preparation 20% Triton X-100 2.5ml 0.5% 500mM EDTA-Na 0.2ml 1mM Distilled water was added to make up to the final volume of 100ml. UNIVERSITY OF IBADAN LIBRARY 167 F. 2nd washing: medium Stock Solution Volume used Finalin Buffer Concentration preparation 100mM HEFES pH 7*4 10ml 10mM 5% Sodium Azide 4ml 0.02% 100mM CaCl2 0.1ml O.lmM 1M NaCl 13ml 130mM Distilled water was added to make up to the final volume of 100ml UNIVERSITY OF IBADAN LIBRARY 168 Procedure The solubilization and purification of Ca 2+ -ATPasewwas performed at 4 oC, except where indicated and was carried out using the procedure of Niggli et. al (1981). 20-100ml of white calmodulin- depleted erythrocyte membrane suspension (150-200mg protein) were concentrated by centrifugation at 36,000 rpm for 15 mins in a Beckman Centrifuge. The pellet was diluted with Buffer A and slowly the required amount of Triton X-100 to a final concentra­ tion of 0.45% was added (0.9mg Triton/mg protein). The solubilized membranes were stirred on ice for 10 mins to ensure complete solubilization. The solubilized membranes were centrifuged at 38,000 rpm for 35 mins in order to remove unsolubilized material. The supernatant containing the solubilized Ca^+-ATPase was carefully removed and pasteur pipette that has been rinsed several times with cold solubilization buffer and the volume was noted. Phosphatidyl choline was added to a final concentra­ tion of 0.5 mg/ml and CaCl2 was added up to 0.1mM and glycerol was also added to make a final volume UNIVERSITY OF IBADAN LIBRARY - 169 - of 10%. The calmodulin-sepharose hB column (4-rnl bed volume) was equlibrated with 2 bed volumes of buffer B (flow rate 10ml/hr; SG 3/250). The protein solution was passed at the same velocity. The calmodulin affinity column was washed overnight after the column has been loaded with the solubilized enzyme with buffer B and 100ml of buffer C (I6ml/hr; SG a/500). The Ca^-ATPase was eluted from the column by using about 100ml of the elution buffer (buffer D) and Ca^+-ATPase assay was performed on 0.6ml fraction (5 min/fraction 7ml/hr; SG 1/125). Fractions containing Ca^+-ATPase activity were pooled and MgC^ was added up to 2mM and CaC^ up to 0.05mM. The Ca^+-ATPase was frozen at -70°C until used specifically for SDS-PAGE. The calmodulin-affinity column was treated after each use as follows: 100ml of buffer E containing 0.5% Triton X-100 and 1mM EDTA was passed through the column to remove phospholipids. 100ml of Buffer F was also passed several times to remove non-specifically bound proteins and was stored in this buffer at 4°C until used again. UNIVERSITY OF IBADAN LIBRARY - 170 - 2.7 Purification of the enzyme in the absence of added phosphatidyl choline The solubilization and purification of p o Ca +-ATPase performed at 4- C except where indicated was carried out using the procedure of Niggili et_ al (1981). 20-100ml of white calmodulin-depleted erythrocyte membrane suspension (l50-200mg protein) were concen­ trated by centrifugation at 36,000 rpm for 15 mins in a Beckman centrifuge. The pellet was diluted with Buffer A and the required amount of Triton X-100 to a final concentrations of 0.4-%, 0.8%, and 1% was slowly added (0.8mg Triton/mg protein, 1.1mg Triton/mg protein, 2mg Triton/mg protein) and glycerol was added to a final volume of 20%. The solubilized membranes was stirred on ice for 10 mins to ensure complete solubilization. The solubilized membranes were centrifuged at 38,000 rpm for 35 mins in order to remove unsolubilized material. UNIVERSITY OF IBADAN LIBRARY - 171 Q The supernatant containing the solubilized Ca^+- ATPase was carefully removed with pasteur pipette that has been rinsed several time with cold solubilization buffer. The calmodulin - sepharose 4B column (4ml bed volume) was equlibrated with 2 bed volumes of Buffer B (flow rate 10ml/hr; Sg ^/250). Buffer B always contained the amount of Triton X-100 that was used for a particular solubilization. The protein solution was passed at the same velocity. The calmodulin affinity column was washed overnight with buffer B after the column had been loaded with the solubilized enzyme. The C.-i -ATPase was eluted from the column by using about 100ml of the elution buffer (Buffer C) and C a ^ - A T P a s e assay was performed on 0.6ml fraction (5 min/fraction, 7ml/hr; SG V l 2 5 ) . Fractions containing Ca2+- ATPase activity were pooled and frozen at -70°C until used. UNIVERSITY OF IBADAN LIBRARY - 172 - 2.8 Assay of purified Ca +-ATPase Principle This method is based on the reaction of phosphomolybdate with the basic dye malachite green to form a coloured complex. Reagents 0.5% Malachite green hydrochloride 0.5g of malachite green hydrochloride (Fluka Chemicals, Switzerland) was dissolved in about 80ml of distilled water. The solution was stirred very well and the volume was made up to 100ml with distilled water. 10% Ammonium molybdate in 4N HC1 50g of Ammonium molybdate, (NH^)^ ^ 07^24* A-I^O); (Hopkins and Williams Ltd., England) was dissolved in 500ml of 4N hydrochloric acid (BDH Chemicals Ltd., England). UNIVERSITY OF IBADAN LIBRARY - 173 - 34% Sodium citrate 34g of Sodium citrate (BDH Chemicals, London) was dissolved in about 80ml of distilled water and stirred for some minutes until all have dissolved. The solution was made up to 100ml with distilled water. Lanzetta reagent 0.045% of malachite green hydrochloride was mixed with 4.2% ammonium molybdate in 4N HC1 in the ratio 3:1 and this solution was stirred for 20 minutes and filtered through Whatman 5 and 0.8ml of 20% Triton X-100 was later added per 100ml of the solution. Procedure Phosphate assay technique The release of inorganic phosphate from ATP was measured according to the method of Lanzetta e_t al, 1979* 0.2ml of the assay medium was suspended in an eppendorf tube containing the the storage buffer and this was transferred to UNIVERSITY OF IBADAN LIBRARY - 174 - Rocomat heater at 37°C and. protein sample was added. The reaction was started with 1mM ATP five minutes after incubation at 37°C. 0.1ml of the medium was transferred into a test tube containing 1.6ml of Lanzetta reagent and mixed vigorously. The reaction was stopped 1 minute after, with 0.2ml of 34% sodium citrate 21^0 (BDH Chemicals, London) and 30 minutes after the reaction was measured at 660nm in a Perkin Elmer Spectrophotometer. The blank contained water, color reagent and citrate. Standard inorganic phosphate (2-10n moles) was prepared using a 1mM K-phosphate solution. 2.9 Binding studies on the interaction of aflatoxin to erythrocyte membrane Principle of emission intensities The model 204 fluorescence spectrophotometer is a grating instrument designed to automatically record fluorescence and phosphorescence emission intensity versus wavelength in the ultraviolet and visible regions for a variety of organic and inorganic materials UNIVERSITY OF IBADAN LIBRARY 175 Fluorescence is the phenomenon whereby a molecule, after absorbing radiation, emits radiation of a longer wavelength. Thus a compound may absorb radiation in the ultraviolet region and emit visible light. This increase in wavelength is known as stoke's shift. At room temperature most organic molecules are in the ground state. Absorption of photons elevates electrons in these molecules to a higher energy state in less than 10 sec - 1 5 After absorption, energy is lost very rapidly by collision degradation (as heat), resulting in the energy of the excited molecules falling rapidly to that of minimal vibrational energy in the lowest excited state. The energy emitted from these molecules in regaining ground state within a period a of less than 10 ” sec. gives rise to a fluorescent peak, showing the stokes shifts Aromatic molecules containing delocalized tT electrons sometimes fluoresce UNIVERSITY OF IBADAN LIBRARY 176 Aflatoxin B-j molecule is a bifurranocou- marin derivative and it has an , B unsaturated lactone ring, and if its irradiated with U.V light it will fluoresce Reagents: Quinine sulphate I0mg/ml lOmg of Quinine sulphate (Hopkins and Williams, England) was dissolved in 1ml of 50mM cone. H2S0^ pH 2. The stock solution was later diluted to the required Odjig/ml concentration. The solution was kept in a brown bottle. lOOmM sodium carbonate buffer pH 10.0 The buffer was prepared by mixing 50ml of 0o1M sodium carbonate (8.1+gNaH CO3/L) and 10.7ml of 0.1N NaOH. The solution was made up to 100ml with distilled water and the pH checked using a pH meter UNIVERSITY OF IBADAN LIBRARY - 177 - 10mM sodium acetate buffer pH 4.0 The buffer was prepared by mixing 41.0ml of 0 .1M acetic solution (5 -7ml /L) and 9 *0ml of 0.1M sodium acetate (8.2g C2Hj02Na/L). The solution was made up to "100ml with distilled water after checking the pH to be 4.0 with a pH meter. Dialysis bag or Visking tubing Visking tubings were purchased from (Scientific Centre, London, U.K). Washing of dialysis bag The visking tubing was rinsed in distilled water followed by boiling in 0.2M EDTA for about three hours. The solution was changed every 50 minutes. The visking tubing was immersed in about 100ml of 0.02M EDTA and stored at 4°C. The dialysis bag was rinsed in distilled water before it was used UNIVERSITY OF IBADAN LIBRARY 178 - Procedure Thoroughly washed tubing was securely tied at one end and checked for leakage. 20>ug of ghost membrane was suspended in 3 -5nil of buffer containing 130mil KC1, 20mM Tris, *5QjuM M g C ^ ) in the tubing and then securely tied at the other end. The dialysis bag was immersed in a beaker containing various concentrations of aflatoxin . The beaker was kept on ice with constant shaking for 6 hours. The free aflatoxin and membrane bound aflatoxin B^ were determined using Perkin Elmer Spectrofluorimeter 204. p 2.10 Proteolysis of Ca +-ATPase Reagents 1mg Trypsin 1mg Trypsin (Boehringer Diagonistics Manheim, FRG) was dissolved in about 800^1 of distilled water on ice and stirred until dissolved. The solution was made up to 1 ml with distilled water and dispensed in 5QM1 aliquots in an eppendorf tube and kept at 4°C. UNIVERSITY OF IBADAN LIBRARY - 179 - Imp; Trypsin Inhibitor 1mg soybean trypsin inhibitor (Boehringer Diagonistics, Manheim, FRG) was dissolved in about 800pl of distilled water and stirred until dissolved. The solution was made up to 1 ml, dispensed in 50pl aliquots and stored at 4°C. 10mg Leupeptin 50mg of Leupeptin/Sigma Chem. London) was dissolved in about 8ml of distilled water and mixed vigourously for it to dissolved. Distilled water was added to make 10ml. The solution was stored in a reagent bottle and stored at 4°C. 2mg Aflatoxin 5mg of aflatoxin B^ (Sigma Chem. London) was dissolved in 10ml of dimethylsulfoxide (DMSO). The solution was stored in a brown bottle at 4°C. Procedure The ATPase samples (0.05icg/ml) were digested on ice with 2ug of trypsin 1 mg/ml. The reaction was stopped by the addition of a 2 fold weight excess of soybean trypsin inhibitor. In order to UNIVERSITY OF IBADAN LIBRARY 180 obtain appreciable amounts of 90 KDa and 76 KDa fragments, the proteolysis was performed in an EDTA storage buffer (130mPi KC1, 20mM H E P E S , pH 7.2 0.05% Triton X-100, 0.05% phosphatidyl choline (PC), 2mM EDTA and 1mM MgCl2) for 5-7 and 90 min respectively. SDS-polyacrylamide Gel electrophoresis was carried out as on page 189• The assay of the ATPase was carried out in the presence of 1/ig aflatoxin B/|. The activity was determine according to the procedure of Lanzetta et al, 1979 on page 173. Calpain digestion was performed in the presence of calpain for 2 hours (1 Iu Calpain per 10/ig ATPase) in the presence of 0.5mM total Ca^* with 20nM calmodulin. Proteolysis was stopped by adding 10 fold molar excess of leupeptin. Assay of the ATPase was done in the presence of 1jig aflatoxin and the activity was determined as on page 173. 2.11 Electrophorectic separation of membrane-bound and purified proteins on continuous gradients of sodium dodecyl sulphate-polyacrylamide gels A very sensitive method for characterizing and UNIVERSITY OF IBADAN LIBRARY 181 - ascertaining the purity of a purified protein is by electrophoresis on a polyacrylamide gel. Electrophorectic procedures are rapid and detection of the polypeptide bands in the gel by staining or autoradiography is sensitive. Electrophorectic migration rates have a fairly predictable relation to the molecular weight of proteins, if the proteins are dissociated and denatured with sodium dodecyl sulphate (SDS) before and during electrophoresis (Weber and Osborne, 1969). Principle Membrane proteins are solubilized by treatment with SDS, which binds to proteins, conferring on them a net negative charge. Most proteins bind a fixed amount of SDS per amino acid, and as a result, proteins acquire a fixed charge to mass ratio and differ only in their size. As the polypeptides become saturated with SDS, they change into helical rods surrounded by an SDS-shell • Electrophoresis of such proteins separates them almost strictly according to size. UNIVERSITY OF IBADAN LIBRARY 182 The mobility of the proteins through a polyacryla­ mide gel is inversely proportional to the logarithm of the molecular weight of the polypeptide. In ordertto ensure that the proteins are unfolded and separated into monomers, the disulphide bonds in them are reduced with dithiothreitol or mercaptoethanol followed by a brief exposure at 100°C. The polyacrylamide is formed by the co- polymerization of acrylamide and N,N-methylene bisacrylamide in the presence of a catalyst (e.g. ammonium persulphate) and an initiator (e.g. ( dimethyl aminopropionitrite or N,N,N' N'-tetra methylethylenediamine). Concentional means of visualizing proteins on electrophoresis gels is by staining using agents like Nigrosin in acetic acid or trichloroacetic blue - zinc sulphate-acetic acid, periodic acid oxidation, followed by treatment with Schiff's reagents (forglycoproteins) etc. UNIVERSITY OF IBADAN LIBRARY Reagents (i) Stock A; $0% w/v Acrylamide, 0.8% w/v Bisacrylamide 30g of acrylamide (Sigma Chemical Co., London) and 0.8g of N,N'-methylene-bisacrylamide (BIO-RAD Labs. California, U.S.A.) were dissolved in 100ml of distilled water in a 100ml standard volumetric flask. The solution was filtered and stored at 4°C. (ii) Stock C; (Separating Gel Buffer) 1.5M Tris HC1 pH 8.8 I8.17g of Tris (hydroxymethyl) aminoethane, base (Sigma Chemical Co., London) was dissolved in about 90ml of distilled water in a beaker and the pH was adjusted to 8.8 with 1M hydrochloric acid. The solution was quantitatively transferredinto a 100ml standard volumetric flask and made up to the mark with distilled water. The solution was stored in a reagent bottle at 4°C. (iii) Stock E: (Stacking Gel Buffer): 1.25M Tris pH 6.8 1 5 .15g of tris (hydroxymethyl) amino methane base (Sigma Chemical Co., London) was dissolved in UNIVERSITY OF IBADAN LIBRARY 184 about 90ml of distilled water in a beaker and the pH was adjusted to 6.8 with hydrochloric acid (1M). The solution was quantitatively transferred into a 100ml standard volumetric flask and made up to the mark with distilled water . The solution was stored in. a reagent bottle at 4°C. Stacking Gel Buffer B : 0.5M Tris-Cl, 8mM EDTA, 0.4% SDS, pH 6.8 3.03g of Tris (hydroxymethyl) amino methane base (Sigma Chemical Co., London), 149mg of sodium salt of (ethylenediaminetetraaceticacid) EDTA (Sigma Chemical Co., London) and 0.2g of sodium dodecyl sulphate, SDS (BIO-RAD Labs. California USA) were all dissolved in about 30ml of double distilled water and the pH was adjusted to 6.8 with 2M HC1. The solution was quantitatively transferred into a 50ml standard volumetric flask and made up to the final mark with double distilled water. The solution was filter through 0.4jiM filter and was later stored in a reagent bottle at 4°C. UNIVERSITY OF IBADAN LIBRARY - 185 - Ammonium Persulphate: 0.75% and 10% 0.0?5g and 10g of ammonium persulphate, (NH^)2 S2O8 (BIO-RAD Labs. California U.S.A.) was dissolved in 10ml of distilled water respectively. The solution was usually prepared fresh. N, N, N', N 1-tetramethyl ethylene diamine (TEMED) This reagent was purchased from Sigma Chemical, Co., Ltd., London and was stored in a refridgerator. Running gel buffer; 1.5M Tris-Cl , 8mll EDTA O. 4% SDS, pH 8.8 45.5s Tris (hydroxymethyl) aminomethane base (Sigma Chemical Co., London), 745mg sodium salt of ethylenediaminetetraaceticacid EDTA (Sigma Chemical Co., London) and 1g of sodium dodecyl sulphate SDS (BIO-RAD Labs. California U.S.A.) were all dissolved in about 150ml of double distilled water and the pH was adjusted to 8.8 with 2M HC1. The solution was quantitatively transferred into a 250ml volumetric flask and was later filtered through,0.45jiM filter and was later stored in the refridgerator. UNIVERSITY OF IBADAN LIBRARY 186 - IPX-Stock Running Buffer 30g of Tris (hydroxymethyl)aamino methane (Sigma Chemical Co., London), 144.ig of glycine (Sigma Chemical Co.,London), and 10g of sodium dodecyl sulphate (SDS), were dissolved in water and made up to the mark in a 1 litre volumetric flask, The solution was stored at room temperature in a reagent bottle. 5X-Stock Electrode buffer pH 8.3 150g of Tris (hydroxymethyl) aminomethane (Sigma Chemical Co., London), 720g of glycine (Sigma Chemical Co., London), 25g of sodium dodecyl sulphate (SDS) (BIO RAD Lab. California USA) and I2g of sodium salt of ethylenediaminetetraaceticacid (Sigma Chemical Co., London) were dissolved in a double distilled water and made up to the mark in a 4 litre volumetric flask (the pH of the concen­ trated solution is slightly higher but it will change upon dilution to the right value. The pH should not be changed with HC1, since chloride anions in the electrode buffer do not allow the formation of a thin band during electrophoresis. UNIVERSITY OF IBADAN LIBRARY 187 - Working Running Buffer The 10X stock running buffer was diluted ten times (10X) just before use by adding 150ml of the 10X stock to 1350ml of distilled water to produce a 1.5 litre solution. Also the 5X electrode buffer was diluted 5 times (5X) by adding 1 litre of the 5X stock to 4 litres of double distilled water to produce 5 litre solution. This is stored at room temperature until it is finished. Stock D (Anionic Detergent Solution) 10% SDS 10g of sodium dodecyl sulphate, SDS (BIO-RAD Labs. California, USA) was dissolved in 100ml of distilled water and the solution stored at room temperature. Sample buffer This was prepared by mixing 1ml of Stock E, 4-.0ml of Stock D, 2.0ml of glycerol (BDH Chemicals Ltd., England),1.0ml of B-mercaptoethanol (Sigma Chemical Co., London), 0.2ml of 2% Bromophenol blue (BDH Chemicals Ltd., England) and 11.80ml of double distilled water to make a total volume of 20.0ml. The solution was stored at 0°C in 1ml aliquots. UNIVERSITY OF IBADAN LIBRARY 188 - Fixing Solution: 10% TCA-50% Methanol I0g of Trichloroacetic acid BDH Chemicals Ltd., England), was dissolved in a little double distilled water. 500ml of methanol (BD Chemicals Ltd., England) was added, and the solution made up to 1 litre with double distilled water. Stain Solution: 0.25% Coomasie Brilliant Blue, 50% Methanol, 7»5% Acetic Acid 0.5g of Coomasie Brilliant Blue (Serva Feinbiochemica, Heilderberg), was dissolved in 100ml methanol (BDH Chemicals Ltd., England). 15ml of glacial acetic acid (BDH Chemicals Ltd., England) was then added and the solution made up to 200ml with distilled water and stored in a brown bottle. I Destaining Solution: 20% Methanol, 7*5% Acetic Acid 4-OOml of methanol (BDH Chemicals Ltd., England) and 150ml of glacial acetic acid (BDH Chemicals Ltd., England) were measured into a 2 litre standard flask. Distilled water was then added up to the 2 litre mark. UNIVERSITY OF IBADAN LIBRARY 189 - Procedure A Making the Gel Sandwich Two glass plates (18 x 16 x 0.3cm each), spacers and combs were properly washed and cleaned with ethanol to ensure removal of grease which might interfere with gel polymerisation. Grease was sparingly used in the assembly of these materials. The grease was first applied to the spacers and the rubber gasket for the casting stand. A sandwitch of the two glass plates was made by using the greased spacers and clamps. The sandwitch was then clamped into the casting stand with the rubber gasket to form a mould for the gel. Adequate care was taken to ensure that minimum grease enters the space where the gel will be formed since grease retards gel polymerization. Deionized water was then carefully poured into the gel space to test for any leakage. The running and stacking gels were prepared as shown in Table ih. UNIVERSITY OF IBADAN LIBRARY - 190 - TABLE 1U Preparation of running and stacking gels (A) Eunning Stacking gel gel 20% 5% Stock A 11.53ml 2.83ml 2.66ml Stock C 4.25ml 4.25ml - Stock E - - 5.28ml Water - 9.18ml 11.22ml Stock D 0.1 7ml 0.17ml 0.22ml Glycerol 0.98ml 0.23ml - TEMED 0.01ml 0.005ml 0.028ml 0.75% Ammonium persulfate 0.26ml 0.335ml — 1.5% Ammonium persulfate 0.57ml After stirring, 1ml of solution was removed from each of the 20 % and 5% Running gels. UNIVERSITY OF IBADAN LIBRARY - 191 - The gradient maker was used for the purpose of mixing the running gel. A long rubber tubing was connected to the free end of the outlet of the gradient maker and served to fill the glass sandwitch with the gel mixture. The gradient maker was placed on a magnetic stirrer, its mixing chamber containing a small magnetic stirrer was filled with 16ml of the 20 % solution while the reservoir chamber was also filled with 16ml of the 5% solution. While stirring, the value of the gradient maker was opened to allow the 3% solution to flow steadily into the mixing chamber. Simultaneously the free end of the rubber tubing was placed between the glass plates of the sandwitch. When all the gel has run into the glass sandwitch, the top of the gel was layered with water. After the gel has polymerized, the water was poured off and the surface of the gel rinsed with distilled water. A comb was inserted and the stacking UNIVERSITY OF IBADAN LIBRARY gel solution was carefully layered on the running gel using a pasteur pipette. After the stacking gel has polymerized, the comb was removed and the gel sandwitch was filled to the underside of the upper buffer chamber using the rubber gaskets. The upper buffer chamber was filled half-way with the working running buffer; the lower buffer chamber of the electrophoresis unit was also filled to about one quarter full with the same buffer. The gel sandwitch sealed to the upper chamber containing the buffer .was lifted off the casting stand and lowered into the lower buffer chamber. Procedure B ' Making the Mini gel Sandwitch Two glass plates (5cm x 4cm x 1.5mm each) spacers and combs were properly washed and cleaned with ethanol to ensure removal of grease which might interfere with gel polymerisation. A sandwitch of the two glass plates was made by using the spacers and clamps. The sandwitch was then clamped into the cashing stand with the gel. Deionized water UNIVERSITY OF IBADAN LIBRARY - 193 - was then carefully poured into the gel space to test for any leakage. Thesrunning and stacking gels were prepared as shown in Table 15* The running gel was poured into the gel assembly to a level of about 3cm below the maximal filling level. The gel was immediately overlay with % diluted running gel buffer. The gel was allowed to polymerise, water was poured on the gel to rinse the gel, the water poured off and stacking gel was poured and the comb was immediately inserted. After about one hour the comb was removed and the gel was clamped to the electrophoresis chamber. Both chambers were filled with electrode buffer, with a syringe; bubbles that were found below the gel were removed. Sample Application and Electrophoresis The protein or membrane samples for electro­ phoresis were prepared by mixing 20pil to 50pl of the sample with 150jil - 180pl of the sample buffer in eppendorf tubes. These were incubated in a UNIVERSITY OF IBADAN LIBRARY 194 TABLE 15 Preparation of running and stacking mlnloels spacers Stacking Running gel' 4-% gel 7% Stock A (ml) 0.250 1.200 Buffer (ml 0.500 1.250 pH 6.8 pH 8.8 teked 5*1 5*1 10% Ammonium persulfate 25*1 25*1 Total Volume 2ml 5ml UNIVERSITY OF IBADAN LIBRARY - 195 - boiling water for 3 minutes to ensure breakage of the disulphide bonds in the proteins. Equal amounts of the samples (40jig - 5Qjug) were then carefully applied to the bottom of the sample wells with a Hamilton syringe. The electrophorectic ■unit was then connected to the power pack unit. Electrophoresis was run at a constant voltage of 60V and a constant current of 25mA in an air- conditioned room until the tracking dye was about 1cm from the bottom of the gel. After electrophoresis, the gel was carefully removed from the slabs, fixed in a solution containing 50% methanol, and 7«5% acetic acid for about 50 minutes and then . stained for about 5 hours with Coomasie Brilliant blue (R250) solution (0.25% Coomasie Brilliant blue, 50% CHjOH and 7«5% acetic acid). The gel was destained by rinsing it several times in a solution of methanol*acetic acid: water (10:7:83 v/v). The gel was later drained and mounted on a glass plate for photography (Plate 1 and 2) UNIVERSITY OF IBADAN LIBRARY - 196 - A B Plate 1; SDS-polyacrylamide gel electrophoresis of erythrocyte purified Ca2+-pumping ATPase from erythrocyte (B). (A) are values (in KDa) indicative of the molecular weights of the protein. The purified enzyme was applied to a 4-7% continuous gradient gel as described in page 194. UNIVERSITY OF IBADAN LIBRARY - 197 - 200 - 116- 9 7 - OJ 66 - Q 45 - 31- 14- Plate 2: SDS-polyacrylamide gel electrophoresis of erythrocyte ghost membrane. The ghost membrane was applied to a 9-20% continuous gradient gel as described on pp. 190 values above are indicative of the molecular weights of the protein markers. UNIVERSITY OF IBADAN LIBRARY - 198 - 2;12 Silver staining technique Principle The sensitivity of silver staining method is about 50 times greater than with Coomassie blue. It is possible to dectect with this method as low as 5ng of protein. Silver staining technique involves about 5 different steps. The first step involves fixing of the protein in the gel with methanol and acetic acid. This step is needed in order to prevent the elution of proteins from gel during the staining process. Methanol removes some of the gel impurity which might interfere with the staining. However, fixing with methanol causes shrinking of gels while the washing of the gel in ethanol and acetic acid allows the gel to swell to normal size, thus helping to remove contaminating buffer and ions (SDS, glycerol and glycine) which might reduce the sensitivity of the method. The third step which involves the use of an oxidizer favours the interaction of silver ions with the protein and also encourages the reduction UNIVERSITY OF IBADAN LIBRARY - 199 - of the bound silver ions to metallic silver during the development step. Albeit, if this step is omitted a negative image of the protein band is obtained and the gel matrix will then be preferen­ tially stained. In addition, the oxidizer enhances the formation of a positive image by converting the protein hydroxyl and sulfhydryl groups to aldehydes and thiosulfate. The development of the colour of the protein bands which is the last step makes use of formaldehyde, a strong reducing agent in alkaline medium. Formic acid so formed in the reaction is buffered by carbonate solution. The alkaline formal­ dehyde reagent reduces silver ions that is bound to the protein to metallic silver. Although, silver ions are also complexed with the polyacrylamide gel, however, their reduction occurs at a much slower rate. Reagents Fixing solution: 50% methanol + 12% acetic acid This was made up by mixing 50ml of methanol (Analytical grade), 12ml of acetic acid and 38ml of UNIVERSITY OF IBADAN LIBRARY 200 - distilled water in a beaker. The solution was stored at room temperature. Washing solution: 10$ ethanol + 5% acetic acid This was made up by mixing 10ml of ethanol (Analytical grade) and 5ml of acetic acid and 75ml of distilled water. The solution was stored room temperature until use. Oxidising solution: 2.5g potassium dichromate K2Gr2°7 + 380jil Q5% H3P0U The oxidizer was made up by dissolving 2.5g of KgCrgOy (Fluka, Chemicals Switzerland) in distilled water and mixed with 38QU1 of 85$ H^PO^ *n a 250ml volumetric flask. The solution was made up to 250ml with distilled water, and stored at i|°C in a brown reagent bottle until use. The solution was diluted 1:10 before use. Staining solution: 5«1g silver nitrate (AgN^) 5.1g of AgNO^ (Pluka, Chemicals, Switzerland) was dissolved in about 200ml of distilled water in a 250ml volumetric flask. The solution was mixed after making it up to 250ml. The solution was stored at UNIVERSITY OF IBADAN LIBRARY 2C1 - The staining solution was diluted 1:10 before use. Developing solution: I9»7g Na2C0^ + 0.5ml 37% formaldehyde I9.7g of Na2C03 (General purpose Reagent (G.R.P.) Hopkin and Williams, Essex, England) was dissolved in about 800ml of distilled water. The solution was kept at room temperature. Before use, 0.5ml of 37% formaldehyde (37ml + 63ml distilled water) was added and stirred for about 10 minutes. The solution was used up immediately or discarded. Never store the solution. Procedure The protein in the gel was fixed for 30 minutes in 150ml solution containing 50% methanol +12% acetic acid and later the protein in the gel was washed twice for 20 minutes in 150ml of 10% ethanol and 5% acetic acid. It was later allowed to stand in the oxidizer for 10 minutes and then was washed 3 times for 2 minutes with distilled water and immersed in the silver staining solution for 20 minutes. UNIVERSITY OF IBADAN LIBRARY This staining solution was later rinsed off with distilled water. The colour was developed "by rinsing the gel with the colour developer until the protein band becomes visible. The developing solution was always prepared fresh. UNIVERSITY OF IBADAN LIBRARY - 203 - CHAPTER THREE EXPERIMENTS AND RESULTS Experiment 1 Investigation of the binding of aflatoxin B/i to erythrocyte membrane It has been shown that aflatoxin binds firmly to at least one site on either bovine or human serum albumin in vitro (Bassir and Bababunmi, 1973)• The effect of functional groups on the interaction of aflatoxin B̂j and with starch, cellulose and seven other derivatives were later studied by Uwaifo and Bassir (1977) using equilibrium dialysis. The observation that the amount of aflatoxin in blood was third highest to that in the liver, 24 hours after injection suggests strongly that aflatoxin could interact with the red cell membrane. The fact that the liver harbours the highest amount of aflatoxin (Wogan, 1968) suggests also that the toxin penetrates the lipid bilayer with considerable ease probably because of its high lipid solubility. UNIVERSITY OF IBADAN LIBRARY 204 In this study, the red cell membrane has been used as a model biological membrane mainly because it is readily available. There is no information in the literature on the consequence of the interaction of the toxin with plasma membrane. This is because the carcinogenic effect of the toxin is believed to be due to its effect on DNA synthesis. Nevertheless, the toxic effect of the toxin on the membrane can not be under-estimated since this may be the primary event that takes place in aflatoxin B/j hepatocellular carcinogens. The aim of the study was therefore, to establish whether or not aflatoxin binds to the red cell membrane. Procedure (A) 80 ̂ g of aflatoxin B^ was preincubated with 24mg of erythrocyte membrane on ice. The aflatoxin B^-membrane complex was washed several times with 100 volumes of a buffer containing l30mM KC1, 10mM Tris, 50 ̂ lg CaCl2 ,and 500 MgCl2 and centrifuged at 16,000rpm for 30 minutes. UNIVERSITY OF IBADAN LIBRARY - 205 - The fluorescence of the resulting solution was measured in a fluorescence spectrophotometer against a blank containing 100pl of untreated membrane. To obtain pH 4 and 10, 100mM acetate buffer and carbonate buffer were used, respectively. The incubation was also performed at 37°C. The exciter and analyzer wavelength used were those previously established for aflatoxin (370nm i.e. exciter, 460nm analyzer wavelength). Quinine sulphate dihydrate in 50mM H2S0^ (0.1 ^ig/ml) was used as a fluorescence standard and was irradiated at 365mm and analyzed at 460nm. (B) Various concentrations of aflatoxin B^ prepared in Dimethylsulfoxide (DMSQ) were made in a beaker containing about 150ml of buffer (130mI1 KC1 and lOmM Tris). 20 ug of erythrocyte membrane protein prepared as described on page 143 was carefully transferred into washed dialysis tubing containing 3.5ml of the buffer and tied at the other end. The tubing was suspended in the buffer containing aflatoxin B^ and UNIVERSITY OF IBADAN LIBRARY 206 dialysed with constant shaking as described on page 173 . The bag was untied after dialysis emptied into an eppendorf tube. 3ml aliquot was taken out and its fluorescence measured in a Perkin-Elmer fluorimeter 204. Results The results obtained by shinning UV light on the supernatants of the first three washing steps, after centrifugation at 16,000 rpm, did not indicate the presence of aflatoxin,as no blue fluorescence was seen in the supernatants; whereas the membrane pellet showed an intense blue fluorescence characteristics of aflatoxin. In addition, super­ natants obtained from the washing steps of untreated membranes did not give any blue fluorescence indicating the absence of aflatoxin. Table 16 shows the various amounts of aflatoxin bound to the erythrocyte membrane. It seems clear from the data that aflatoxin still binds firmly to the membrane despite washing the membrane thoroughly. It may be inferredfrom UNIVERSITY OF IBADAN LIBRARY 207 - TABLE 16 Effect of pH and temperature on the binding of aflatoxin to the erythrocyte ghost membrane. AFB^-treated nS AFBibound per jug membrane protein membrane zero min 10 min 30 min Control 0.0 0.0 0.0 pH 4.0 3.12 + 0.10 3.91 + 0.10 4.63 + 0.01 *3.31 + 0.14 *4.21 + 0.13 *4.68 + 0.02 pH 7-4 2.38 ♦ 0.03 3.11 + 0.10 3.76 + 0.03 *2.43 + 0.05 *3.18 +_ 0.02 *3.83 i 0.01 pH 10.0 1.24 + 0.12 1.72 + 0.03 2.31 + 0.03 *1.20 + 0.1? *1.68 + 0.01 *2.30 + o.02 Pellet obtained after incubating the membrane with aflatoxin solution at lj.0C was irradiated at 360nm and analyzed at i+60nm. * Data were obtained from experiment carried out at 37°C Each value is a mean of five different determinations + standard error. UNIVERSITY OF IBADAN LIBRARY 208 the results that aflatoxin binds spontaneously to the membrane even though more binding seemed to take place on longer period of incubation. Similar results were obtained when binding was studied at 37°C provided that incubation was not allowed to proceed beyond 30 minutes. Studies on the effect of pH on the binding of the toxin to the membrane indicate that more of the toxin molecules are bound at acidic pH while the degree of binding is drastically reduced at alkaline pH. Results obtained from binding studies using equilibrium dialysis technique confirm that aflatoxin B/| binds firmly with the cell membrane. Scatchard plot of the data obtained as shown in Fig. 16 indicate that the binding of aflatoxin to the membrane is a non-cooperative process and that 4.5 nmoles of the toxin was bound per ug membrane protein. UNIVERSITY OF IBADAN LIBRARY - 209 - Fig. 1 6: Scatchard plot of the binding of aflatoxin Bi to erythrocyte’ membranes. nanomoles AFB1 pg membrane protein nanomoles AFB~ UNIVERSITY OF IBADAN LIBRARY 210 - Conclusion Aflatoxin B-j binds spontaneously and irreversibly to erythrocyte membrane in a non- cooperative manner. % UNIVERSITY OF IBADAN LIBRARY 211 - Experiment 2 Influence of aflatoxin on erythrocyte membrane Ca 2+ ATPase activity The Ca 2+ -pumping ATPase of the plasma membrane is an integral protein with a Mr of about 136KDa (Niggli et al, 1 981) and representing less than 0.1$ of the erythrocyte total membrane protein (Knauf et al, ^ ^ 7 k ) • It has been suggested that about 700 copies of this protein are present in the erythro­ cyte membrane (Rega and Garrahan, 1975)- The most important endogenous regulator of the protein is calmodulin which becomes active after binding calcium ions (Lin et al, 1974)* The catalytic and the calmodulin functional domains of the protein have been shown to possess important amino acids residues such as lysine (Kretsinger, 1980). Concidentally1 these amino acids have also been shown to be important for the binding of aflatoxin to BSA. It thus seems probable that aflatoxin B-j could inhibit the Ca 2+-pumping ATPase by binding to such amino acid residues, most especially because data reported in experiment I in this thesis UNIVERSITY OF IBADAN LIBRARY - 212 - have shown that, although the toxin binds spontaneously and irreversible to the erythrocyte membrane, the binding is favoured by increasing the acidity of the medium. The aim of this study therefore was to investigate the effect of aflatoxin on the activity of the pump with or without calmodulin. Procedure Haemoglobin-free erythrocyte ghost membranes were prepared from normal fresh erythocytes according to the procedure of Dodge e_t al_ (1963) as described on page 14-3. Membrane protein was determined according to the procedure of Lowry £t al_ (1931) and as described on page 149. Ca2 ~ 4* ATPase was assayed in a medium containing final concentration (100mM HEPES, 2mM KC1, 100mM MgCl2, 1mM CaCl2, 50mM EGTA) with various concentrations of Aflatoxin B^. The activity was assayed in the presence and absence of calmodulin described on page 157 under 'materials and methods'. To study the ATP dependence, various concentrations of ATP were used to start the reaction and aflatoxin B>| concentration with maximum inhibitory UNIVERSITY OF IBADAN LIBRARY - 213 - effect was used (1C)pag aflatoxin). The activity was also assayed as described above. The amount of inorganic phosphate liberated during the ATPase reaction was determined by the method of Fiske and Subbarow (1925) and as described on page 158 under 'materials and methods'. Results Fig. 17 shows the effect of aflatoxin on the basal and calmodulin stimulated enzyme. It can be seen from the figure that aflatoxin has no significant effect on the basal activity of the enzyme. The profile for the calmodulin stimulated enzyme, shows that aflatoxin B^ inhibits the calmodulin stimulated enzyme in a concentration dependent fashion; maximum inhibitory effect of about 50% was obtained with almost 10jig aflatoxin Bi (Fig. 18). Fig. 19 and 20 show the kinetic analysis of the data obtained from studies on the ATP dependence of the inhibition by aflatoxin B^ and they shows that the toxin has no effect whatsoever on the UNIVERSITY OF IBADAN LIBRARY Pig.17: Effect of varying concentrations of aflatoxin B-j on the basal and calmodulin stimulated activity of erythrocyte plasma Ca_I ATPase. In the presence (circles) and abscence (triangles) of calmodulin. UNIVERSITY OF IBADAN LIBRARY 215 ► 1.5 UNIVERSITY OF IBADAN LIBRARY - 216 - 80p i' AFLATOXIN B-(jjg) Pig. 1 8: Inhibition of calmodulin stimulated erythro­ cyte membrane Ca2+~ATPase by aflatoxin . UNIV INHIBITIONERSITY OF IBADAN LIBRARY - 217 - Fig.19J Influence of afla toxin B| on the ATF dependence of erythrocyte membrane Ca^+-ATPase. In the pcrfelseetnocxei n (Bt-rj.iangles), absence (circles) of UNIVERSITY OF IBADAN LIBRARY - 218 - 1.0“ 0.5- 0 T-----1.0 --------------------------------------- 1-2.0 Fig. 20: Influence of aflatoxin on the ATP dependence n of calmodulin stimulated erythrocyte membrane Ca'1'* ATPase. In the presence (triangles) absence (circles) of aflatoxin B^. U (j jCar ATPPia se activity- N moles mg protein i hrIVERSITY OF IBADAN LIBRARY 219 - maximum velocity and affinity of the enzyme for ATP in the absence of calmodulin. In contrast, both maximum velocity (Vmax) and the affinity of the enzyme for ATP (Km (ATP)) were reduced by aflatoxin in the presence of calmodulin by at about 15% and 50% respectively. Thus the toxin has no significant effect on the affinity of the enzyme for ATP even in the presence of calmodulin. Table 1? shows the summary of the Vmax and Km values of erythrocyte plasma membranes. Conclusion Aflatoxin inhibits the calmodulin stimulated activity of erythrocyte Ca^+-pumping ATPase while it has no effect on its basal activity. Whereas maximum velocity of the stimulated enzyme is drastically reduced by aflatoxin B^ there is no significant effect on the Km of the enzyme with or without calmodulin. UNIVERSITY OF IBADAN LIBRARY 220 TABLE 17 Effect of aflatoxin on the Vmax and Km value of the erythrocyte plasma membrane Ca^*-ATPase. Km (umoVlm Paix mgprot -1 h -1 ) OuM ATP) Ca2+ ATPase Erythrocyte 2.2 + 0.10 6600 + 500 membrane (2.4 + 0.20) (6800 + 600) (basal) Ca2+ ATPase Erythrocyte 6.25 + 0.50 400 + 50 membrane (calmodulin) (3.40 + 0.15) (340 7 80) ( ) were obtained in the presence of aflatoxin . UNIVERSITY OF IBADAN LIBRARY 221 - Experiment 3 Inhibition of erythrocyte purified Ca 2+ ATPase by aflatoxin B-j Experiments on the interaction of aflatoxin v/ith membrane bound Ca 2+ ATPase have shown that the toxin reduced the maximum velocity (Vmax) of the calmodulin-stimulated enzyme only, without affecting its basal activity and its affinity for ATP whether or not calmodulin is present. Knowledge on the kinetic properties of the pump has shown that calmodulin increases the Vmax of the pump by increasing its affinity for Ca 2+ and thus increasing its turnover (Jeffery et al, 1981; Larsen g_t al, 1981). Accordingly, the activation has been shown to stimulate both the rate of phosphorylation of the pump and that of its dephosphorylation (Jeffery et al, 1981; Luthra et al, 1980). Since the inhibition of calmodulin stimulated membrane-bound enzyme is not ATP dependent, it seems logical to suggest that the toxin could be interfering with the pump by binding to some amino acid residues in the calmodulin binding domain, which has a sequence with an obvious predominance of basic amino acid residues. UNIVERSITY OF IBADAN LIBRARY 222 - In order to gain insight into the mechanism of inhibition of the pump by aflatoxin Bi, the enzyme has been detergent-solubilized and purified on calmodulin affinity chromatography. It is thus possible to study the interaction of the aflatoxin B-i directly with the pump. The aim of this experiment was therefore to establish the mode of inhibition of the calmodulin-stimulated pump by aflatoxin . Procedure To study the effect of aflatoxin with the purified enzyme, various concentrations of aflatoxin preincubated with 3,Mg purified ATPase in eppendorf tubes containing the enzyme and aflatoxin were then incubated at 37°C in a water bath with constant shaking. The assay medium was added to obtain a final concentration (l20mM KC1, 1mM MgClp, 30mM HEFES, 10uM CaClp, pH 7.h). Calmodulin (2jag) was also added and the reaction was started by addition of 2mM ATP. The reaction was allowed to proceed for 3 minutes. The amount UNIVERSITY OF IBADAN LIBRARY - 223 - of inorganic phosphate liberated was measured by the methof of Lanzetta et_ ad 1979 as described on page 173- The experiment was carried out in duplicate. In addition, series of experiments on the ATP dependence of the effect of aflatoxin were carried out using the concentration of aflatoxin that gave maximum inhibition. ATPase activity was determined as described above and various concentrations of ATP ranging from 0.5-2.5mM were used to initiate the reaction. The experiments were similarly carried out in duplicate. Results Fig. 21 shows the influence of aflatoxin B^ on the basal and calmodulin-stimulated activity of the purified enzyme. It can be seen from the figure that the effects of aflatoxin on the purified erythrocytes are similar to that seen in case of the membrane-bound enzyme. The data show clearly that aflatoxin has no significant effect on the basal activity of the enzyme whereas the stimulated enzyme was inhibited in a concentration UNIVERSITY OF IBADAN LIBRARY 224 Fig. 21: Effect of varying concentrations of aflatoxin E- cn the basal and calmodulin stimulated activity* of the purified ATPase. In the presence (circles) and absence (triangles) of calmodulin. UNIVERSITY OF IBADAN LIBRARY 225 dependent manner by aflatoxin by about 50% with almost 12 jamoles of the toxins. Q?ig. 22^ Analysis of the data obtained from studies on ATP dependence, as shown in rf Fig. 23 and 2k,X l and as summarised in y aflatoxin Bi It is well established that the Ca^+-ATPase of the erythrocyte ghost is remarkably stable in the membrane-bound form, apparently because the lipid surrounding the protein molecules ensures perfect conditions for preservation of the enzyme functional conformation. Solubilization of this enzyme is frequently accompanied by the loss of its hydrolytic activity (Niggli et_ al_, 1979; Niggli et al, 1981); Scharff 1981. The nature of the detergent used for solubilization nothwithstanding, its concentration is an important factor in achieving maximal ATPaseaactivity. Several workers have shown that in the absence of detergent, sonicated lipid dispersions were unable to support ATPase activity whereas activity was observed when detergent and phospholipid were present (Kelson and Hanahan 1985). UNIVERSITY OF IBADAN LIBRARY - 232 - Results of several studies have shown that acidic phospholipids are five to eight times more effective than phosphatidyl choline in promoting activity of the pump by increasing both its maximum velocity and its apparent calcium affinity (Tavema and Hanahan, 1980; Niggli et al 1981; Sarkadi et al, 1982) while causing a significantly lower (Ca ) than calmodulin with no co-operativity in the calcium activation (Enyedi et al, 1987; Sharff 1978; Villalobo e_t al 1986; Enyedi ejt al 1987). It has also been shown that, although phosphatidyl serine stimulates the ATPase activity, it was achieved as a result of the low detergent concentration used, where mixed micelle formation was complete (Taverna and Hanahan, 1980). On the other hand, higher detergent concentration resulted in a decrease in ATPase activity and this might be due to a dilution of the lipid by excess detergent. On subjecting the purified enzyme to tryps inisation, Enyedi et al (1987) were able to deduce that the 8lKDa tryptic fragment is still regulated UNIVERSITY OF IBADAN LIBRARY - 233 - acidic phospholipids while the 76KLa fragment is not, Further studies indicated the actual position of phospholipid binding and was tentatively placed at the N^-terminal 5KDa of the 81-KDa fragment (Papp at ad, 1989)• Zvaritch e_t al (1990) demonstrated that the phospholipid domain has about 44 amino acids residues and it is lysine rich, while Brodin et al (1991) showed that there are possibly two functional domains of phospholipid interaction. In this study, the effect of triton X-100 and acidic phospholipids on the inhibition of Ca‘~f- ATPase by aflatoxin B-j have been assesed, in an attempt to distinguish between the possible site of attack by aflatoxin since acidic phospholipids and proteolysis mimick the effect of calmodulin and act at different sites on the enzyme. Procedure Purified C a A T P a s e was prepared from normal fresh erythrocytes according to the procedure UNIVERSITY OF IBADAN LIBRARY - 234 - described, earlier. Frotein concentration was determined according to a modified procedure of Markewell et_ al (1981). To study the effect of aflatoxin on cardiolipin-stimulated enzyme, various concentrations of aflatoxin B^ were prepared in dimethylsulfoxide (DNSO) and these were added as described earlier. AlTase activity was measured by the procedure of Lanzetta £t_ al, (1979) as also described earlier. In studies on the effect of triton X-100 on the inhibition of the enzyme by aflatoxin B^ various concentrations of triton X-100 were used for solubilization prior to affinity chromato­ graphy on calmodulin column as earlier described. ATPase activity was measured by the procedure of Lanzetta et al (1979) as earlier described after initiating the reaction by addition of ATP solution. UNIVERSITY OF IBADAN LIBRARY - 255 - The effects of varying concentrations of phosphatidyl serine (PS) and phosphatidyl choline (PC) on the inhibition of the pump by aflatoxin were also studied. Similar concentrations of aflatoxin B-i (1 ug) were added to the enzyme (5 ug). Calmodulin (2 ug) was added to further stimulate the enzyme. ATPase activity was determined as described earlier. Results Figs. 25 and 26 shows the effect of aflatoxin B^ on basal and cardiolipin-stimulated activity of the purified enzyme. The figures show that, aflatoxin B^ inhibited the activity of the enzyme in a concentra­ tion dependent manner by about 28% while it has no effect on the basal activity of the enzyme. Effect of aflatoxin B^ on the solubilized and purified enzyme (Fig. 27) indicated that the activity of the UNIVERSITY OF IBADAN LIBRARY 236 5- A 3 ♦ 2 1 A A i - - - - - - A- - - - - - A- T T~ 0 2 5 10 M,moles (AFB^) Fig. 25: Effect of varying concentrations of aflatoxin B-j cn the cardioliptn (DPG) stimulated Ca^+-ATPase. In the presence (circles), absence (triangles) cf cardiclipin. Ca^ATPase activity (u moles Pi mg ProtT1 mm UNIVERSITY OF IBADAN LIBRARY - 237 - Fig. 26: Percentage inhibition of the cardiolipin stimulated Ca^+-ATPase by aflatoxin B-j. UNIVERSITY OF IBADAN LIBRARY 238 - 6 - _ 5- Trixtonx-100 I Protein mg •_ mg Fig. 27: Iton flpureontceei n ofo n vatrhyeing the ratios of triton X-100aflatoxin B^. In tihneh ibpirteisoenn ceo f oft hec aClmao2d+u-lAiTnPase by (closed circles) presence of (squares) calmodulin and aflatoxin B^, presence of (open circles) no calmodulin and aflatoxin presence of aflatoxin (triangles). Caz ATPase activity ( jjm oles Pi mg p rc t“ f min UNIVERSITY OF IBADAN LIBRARY - 239 - enzyme increases with, increase in the ratio of mg triton X-100: mg protein but doubled when the ratio of mg triton X-100: mg protein was increased to 2. However no further stimulation was observed in the presence of calmodulin but stimulation was doubled with 2mg triton X-100/mg protein. The effect of aflatoxin on the calmodulin stimulated enzyme indicated that only enzyme prepared in the presence of 2mg triton/mg protein was inhibited by about 50% while at lower ratio of triton X-100 to mg protein, the toxin has no effect. Aflatoxin has no effect on the basal activity of the enzyme. Fig. 28 shows the effect of aflatoxin B^ on phospholipid activated enzyme. It can be seen from the figure that there is no significant activation of the enzyme by PC alone. Calmodulin stimulated the enzyme in the PC by 4 fold, the calmodulin stimulated enzyme in the presence of PC was inhibited by about 50% by aflatoxin B^. Aflatoxin B^ has no effect on the basal activity of the enzyme in the presence of PC. UNIVERSITY OF IBADAN LIBRARY 240 Fig. 28: Effect of varying concentrations of phosphatidyl choline on the inhibition of the Ca^+-ATPase by aflatoxin (in the presence of: (a) calmodulin (big closed circles); (b) calmodulin and aflatoxin (triangles); (c) absence of calmodulin and aflatoxin B>| (squares) and (d) presence of aflatoxin B^ (small circles)). UNIVERSITY OF IBADAN LIBRARY Fig. 29 shows that phosphatidyl serine (FS) stimulated the enzyme in a concentration dependent manner; maximum stimulation was obtainable with about 8 nmoles of PS. There was no further stimula­ tion of the enzyme by calmodulin; aflatoxin inhibited the PS stimulated enzyme by about 28%. Conclusions Aflatoxin inhibited the cardiolipin stimulated Ca^+-ATPase by about 28%, while it has no significant effect on the basal activity of the enzyme. Triton X-100 maximally activated the enzyme only when the ratio triton: protein is 2. Aflatoxin B^ inhibited the triton activated enzyme by about 50%. PC alone did not activate the pump. Calmodulin- stimulated enzyme in the presence of PC was inhibited by aflatoxin B^. In addition aflatoxin B-j inhibited 'the PS stimulation of the enzyme by about 28% while the enzyme was not further stimulated by calmodulin. In conclusion, aflatoxin B^ has effect only on the stimulated enzyme and inhibition depends on the type of activator used. UNIVERSITY OF IBADAN LIBRARY 242 Fig. 29: Effect of varying concentrations of phosphatidyl serine on tlie inhibition of the Ca^+-ATFase by aflatoxin B^ (in the presence of; (a) calmodulin (big circles); (b) phosphatidyl serine (triangles; (c) aflatoxin (small circles)). ATPase ac t iv i ty UNIVERSITY OF IBADAN LIBRARY 243 - Experiment 5 Interaction of aflatoxin with partially proteolysed purified C a + ATPase It has been shown that either of chymotrypsin and trypsin will activate the membrane-bound C a r t ATPase by partial proteolysis. This activation mim'icks the effect of calmodulin leaving the activated pump calmodulin insensitive (Benaim e_t al, 1984). Several studies have shown that the steady state level of the phosphorylation is reduced when the enzyme is treated with trypsin in the absence of Mg“ + (Zurini et al, 1984). Studies on the proteolysis of the Ca^— pump by Zurini et al, (1984) showed conclusively that the fragments of 90KDa, 85KDa, 81KDa and 76KDa are produced on partial proteolysis. There is also a removal from the enzyme of a fragment with a molecular weight of 'v 33KDa (Zurini et al, 1984). It has been shown that the 90KDa fragment still contains the calmodulin binding domain (Enyedi et al, 198?) and thus responds normally to calmodulin/ while the 85KDa fragment is UNIVERSITY OF IBADAN LIBRARY 244 is less responsive to calmodulin^indicating that the calmodulin binding domain is not completely removed by proteolysis in this fragment (Zvaritch et al, 1990). Studies on the 76KDa and 81KDa fragments show that they are no longer responsive to acidic phospholipids and calmodulin. Altogether these findings indicate clearly^that acidic phos­ pholipids and calmodulin interact with the ATPase at two different sites. It is clear that the effects of trypsin and chymotrypsin have no physiological meaning whereas the effects of the intracellular Ca^+ dependent cysteine protease> otherwise called calpain could be physiologically relevant (Bond and Clough, 1972; James e£ al, 1989; Wang at al, 1988). This protease has been shown to increase the basal activity of the pump in the erythrocyte membrane but does so much slowly than the extracellular proteases, reaching the maximal levels attainable with calmodulin in one to two hrs. It has also been shown that the treatment with calpain reduces UNIVERSITY OF IBADAN LIBRARY - 245 - the apparent molecular weight of the enzyme by l2KDa, leaving in the membrane, a component of apparent molecular weight of 124KDa fragment, which is insensitive to calmodulin (Zurini e_t al, 1984; Zvaritch e_t al, 1990). Its seemsppertinent therefore to study the interaction of aflatoxin on the partially proteolysed Ca^ -pump, so as to assess the possible site of interaction of the toxin on the Cac+-pump. Procedure Calpanized ATPase was prepared from normal fresh erythrocytes according to the procedure described earlier. Protein concentration was determined according to the modified procedure of Markwell elfc al (1981) as described earlier. Various concentrations of aflatoxin were preincubated with 3ug of the enzyme in eppendorf tubes for 50 minutes on ice and transferred to a water bath at 57°0. ATPase activity was estimated in the absence and presence of calmodulin according to the procedure of Lancetta as described earlier. UNIVERSITY OF IBADAN LIBRARY 246 - In experiment on the influence of aflatoxin B/j on trypsinised ATPase, the enzyme was subjected to trypsinisation for 5-7 niin and 90 mins respecti­ vely in order to obtain the 90KDa and 7&KDa fragments. The trypsinised enzyme (50ug) was preincubated in an eppendorf tubes on ice with aflatoxin B-i (10 ug) for about 50 mins and transferred to a water bath at 57°0. The assay buffer were added to a final concentration of (I20mil KC1, 1mM MgC^, 10uW CaCl2 t 30mM HEPES, pH 7«4), the ATPase activity was determined in the presence and absence of calmodulin as previously described under 'materials and methods' page 173 . Results Table 19 shows the effect of aflatoxin B/j on calpanised ATPase. The Table shows that varying concentrations of aflatoxin have no effect on the activity of the partially proteolysed enzyme in the presence and absence of calmodulin. There was no stimulation of this enzyme by calmodulin. UNIVERSITY OF IBAD N LIBRARY - 24? - Plate 3: SDS-polyacrylamide gel electrophoresis of partially purified Ca^-pumping ATPase from erythrocyte. (A) are values 4(in X10- Da) indicative of the molecular weights of the protein markers (B) is the purified enzyme. (C) is fragment for 5 mins. (D) is fragment for 15 mins. (D) is fragment for 30 mins. (E) fragment for 1 hr. The partially purified enzyme was applied to a 4-7% continuous gradient gel as described on page 194. UNIVERSITY OF IBADAN LIBRARY 248 - TABEE 1 ? Effect of aflatoxin B1 on calnanised ATPase (Aflatoxin) Specific Activity jumoles Pi mg prot min'-1 (jumoles) -Calmodulin + Calmodulin 0 2.2 + 0.01 2.5 2.1 + 0.02 2.3 + 0.02 k . 9 2.3 + 0.C1 2.5 + 0.01 l .Ur 2.6 + 0.01 2.7 ± 0.01 12.3 2.8 + 0.02 2.8 + 0.03 Each value is a mean of five different determinations + standrd error. UNIVERSITY OF IBADAN LIBRARY oT “ o• + l o• CM - 24-9 - Table 20 shows the effect of aflatoxin on the trypsinised enzyme. According to the Table the enzyme fragment produced after 5 minutes which is 90KDa was calmodulin sensitive and was inhibited by aflatoxin by about 50%. The 76KDa fragment produced after 90 minutes of incuba­ tion with trypsin was calmodulin insensitive. The ATPase activity of this fragments could not be inhibited by aflatoxin B̂| even at the fragment that give maximum inhibition. Conclusion Aflatoxin B^ has no effect whatsoever on the calpanised enzyme, whereas the toxin inhibited the ATPase activity of the enzyme partially trypsinised for 5 (which correspond to the 90KDa fragments) the toxin has no significant effect on the enzyme trypsinised for 90 which corresponds to the 76KDa fragments of the ATPase. UNIVERSITY OF IBADAN LIBRARY 250 - TABLE 20 Effect of aflatoxin B/i on -partial proteolysis of the Ca^i- ATPase by Trypsin Period of jumoles Pi mg prot”— ̂ min— "1 incubation (min) -Calmodulin + Calmodulin 0 1.00 + 0.01 4.50 + 0.05 (Aflatoxin B,,) (0.86 + 0 .01) (2.50 + 0 .01) ? 1.19 ± C.02 4.10 + 0.05 (Aflatoxin B^) (1.05 + 0.01) (2.19 + 0.07) 901 4.65 + 0.06 4.54 + 0.01 (Aflatoxin B>|) (4.40 + 0.01) ( ) in the presence of aflatoxin B^ Each value is a mean of five different determinations + stand error. UNIVERSITY OF IBADAN LIBRARY - 251 - CHAFTER FOUR DISCUSSION A negligible amount of C eT+ is contained in the extracellular and intracellular fluids; most of the calcium of higher organisms is immobilized in the bones and teeth as hydroxy­ apatite Ca^Q(PO^)g (0H)2 • Calcium concentration in the extra cellular compartment including blood plasma is regulated by the mobilization of Ca^+ from and to bone deposits, and is fixed at about 3mM; approximately half of this amount of Ca2+ is in the ionized form. Extracellular or plasma Ca^+ is important from the stand point of its relation- ship to the intracellular Ca^+ , because the cytosolic calcium performs the fundamental role of transducing signals to a large number of biochemical processes in the various subcellular compartments the link between extracellular Ca^* and the signalling Ca^+ inside the cell is not well under­ stood, but it seems clear that the extracellular pool provides a relatively large reservoir from which Ca + is drawn and made to flow into the cell. UNIVERSITY OF IBADAN LIBRARY - 252 - The maintenance of the extracellular CaT+ within a narrow concentration range ensures that a constant source of Ca^* will always he available to cells. Despite the very large inwardly directed Ca^2 + gradient across the plasma membrane, the background concentration of a free Ca^+ in most cytosols oscillates indeed between 0.1 and 0.2)iM (Carafoli, 1987). The essential concept of cellular Ca^+ homeostasis (Carafoli, 1987) centers therefore on the ability of certain components of the membrane to perform the task of decoding the information of Cs l * and of controlling its cytosolic concentration. These membrane components consist of a large number of specific C & + binding protein soluble or insoluble to the membrane. It follows therefore that in the presence of a large Ca^+ pressure, even very minor changes of this permeability would result in significant swings in the intracellular concentration of free Ca 2+ and would thus efficiently influence the UNIVERSITY OF IBADAN LIBRARY - 253 - modulation of Ca^+ targets. In case of a breakdown of the permeability barrier of the plasma membrane, cellular calcium overload would unavoidably occur and this could result in cell death. It is well established that flooding of the cytosol with Ca + is indeed a frequent and early event in cell pathology (Niggli, Adunyah, Cameron, Bababunmi and Carafoli, 1982; Olorunsogo, Okudolo, Lawal, Falase, 1985). It seems clear therefore that eucaryotic cells have chosen to survive in a permanent condition of controlled danger, a dynamically convenient but nevertheless perilous choice where the line separating cells from Ca^+ poisoning may at times be very tenuous. Clearly the need for systems extruding Ca^+ from cells to cancel its downhill influx is glaring. Although eucaryoti< cells generally satisfy most of their Câ + demands q 0 by extracting Ca^+ from internal Ca^+ stores, it is very clear, that long-term maintenance of the Ca^+ gradient across the plasma membrane is the result of tl UNIVERSITY OF IBADAN LIBRARY - 254 - concerted operation of the importing system (the Ca^ + channel) and of two exporting systems (the Ca^+-pump and the Na+/Ca^^-exchanger) of the plasma membrane. These transport systems have different kinetic properties, poised to satisfy the different requirements of celjlf s during their functional cycle (Carafoli, 1987). Indeed, there will be situations where Ca^+ must be regulated in the cytosol, or in other cell compartments, very rapidly and with utmost precision e.g. the contraction- relaxation cycle of muscle especially fast muscles. Conversely other situations may require slower movements of bulk amounts of Ca^+. The systems mentioned above are diversified to do Just that, since they have different affinities of interaction with Ca^+, and different total C a h a n d l i n g capacity. In general, whenever the need arises to transport Ca + with high interaction affinity, ATPases are chosen, since this appears to be the only transport mode that confers to the system high Ca + affinity. As a result, cells rely UNIVERSITY OF IBADAN LIBRARY - 255 - solely on ATPases for the fine tuning of their C a O n the other hand, more options are open to situations that demand the movement of hulk amounts of Ca^+ with intermediate affinity. Exchangers, channels and electrophoretic uniporters are all low Ca^+ affinity systems. However, in red cells the major extrusion mechanisms is the Ca^+-ATPase (Schatzmann, 1966). The availability of a specific inhibitor for the pump has several implications and these include the ease with which experiments on the evaluation of the physiological and pathological role of the pump will be designed and the application of the chemistry of such substance to the development of a drug which may find particular use in overcoming heart failure. Although, aftatoxin effects its carcinogenic action by intercalating with DNA through H^-guanyl residue to form an adduct, the effect of its direct interaction with membrane components is still unknown. It seems however, reasonable to surmise that since aflatoxin binds UNIVERSITY OF IBADAN LIBRARY - 256 - amino acid residues of serum albumin (Bassir and Bababunmi, 1973) and certain functional groups of polysaccarides (Uwaifo and Bassir, 1977) it seems likely the toxin could bind to the red cell membrane components with or without modifying the properties of the membrane. It is based on this reasoning therefore, that the toxin has been used as a tool to inhibit the pump. It is convenient to use red cell membrane as a model for two main reasons. Firstly, red blood cells are readily available in sufficient quantities and secondly, they have no nucleus and no organelles, hence uncontaminated haemoglobin-free erythrocyte ghost membranes are readily obtained from these cells. In order to determine whether or not aflatoxin binds to the red cell membrane, advantage has been taken of the fluorescent properties of the toxin. The finding that aflatoxin B^ binds sponta­ neously and irreversibly to red cell membrane (Table 16) suggests that the toxin interacts with certain components of the membrane. Although, certain functional groups have been found to be UNIVERSITY OF IBADAN LIBRARY - 257 - necessary for the binding of the toxin to proteins and to carbohydrate (Uwaifo and Bassir, 1977), it is not known if the same groups are involved in the binding of the toxin to the red cell membrane. It seems likely, however, that groups such as the amino and carboxyl groups of either amino acid residues of proteins or other groups on the lipid and carbohydrate components of the membrane could possibly be involved in the binding. Depending on the number of aflatoxin molecules that bind to the membrane, there is the possibility that spontaneous and irreversible binding of the toxin to the membrane could cause pertubation of the membrane and also disrupt the assymetry of the phospholipids and fluidity of the membrane. It is also possible that since the toxin is lipid soluble it could trasverse the membrane and subsequent] bind to a lipid soluble component on the cytoplasmic face of the membrane. Data obtained from studies on the temperature and pH dependence of the binding of the toxin to UNIVERSITY OF IBADAN LIBRARY - 258 - the membrane reveal that more binding occurred at acidic pH 4.0, indicating that the binding of the toxin is enhanced by the degree of protonation of the groups or amino acid residues responsible for binding the toxin to the membrane. It seems likely that protonation of the membrane could affect the conformation of the various components of the membrane in such a way that more toxin molecules are bound. The results also show that there was a reduction in the binding of the toxin to the membrane at alkaline pH (pH 10). Since coumarins are labile at alkaline pH, due to hydrolytic cleavage of the unsaturated lactone ring of the coumarin nucleus (Fujimoto and Ohba, 1975) there is thus the possibility that there will be a reduction in the fluoresence of aflatoxin at pH 10. Using equilibrum dialysis technique the amount of aflatoxin bound to the membrane was evaluated spectrofluorimetrically. Scatchard plot of the data obtained (Fig. 16) revealed that 4.5nmoles of aflatoxin B>| bind per ng membrane UNIVERSITY OF IBADAN LIBRARY - 259 protein. The implication of this finding is that the binding of the toxin to the membrane is likely to be to specific groups on the membrane. Furthermore, it seems that if such a small amount of the toxin binds to the membrane it could be binding to a component of the membrane that has a very low concentration in the membrane. The fact that Ca^+ is the most preponderant cation in the human body and the observation that its concentration is carefully regulated in the cell interior, despite the passive influx of this cation into cells and its function in the regula­ tion of metabolism, suggest that aflatoxin binding to the membrane could cause a change in the permeability of the cell membrane to the cation in addition to modifying its efflux mechanism. Since the cell membrane is generally permeable to calcium, it appears that aflatoxin effect would be more drastic or pronounced on the efflux mechanism. The studies reported in this thesis cente: mainly on the effect of the toxin on membrane-bound and purified erythrocyte Ca^+-ATPase. UNIVERSITY OF IBADAN LIBRARY 260 Experiments on the membrane-hound enzyme should yield information on the effect of the toxin on the pump protein in its natural environment, while studies conducted on the purified enzyme are relevant from the perspective of the exact mechanisms of interaction of the toxin with the enzyme protein. Architectural models of the secondary structure of the plasma membrane calcium pump have been predicted and developed by simple analogy with the structure of the sarcoplasmic reticulum Ca +-ATPase and on the basis of the homology in the amino acid sequences of the two pumps (Shull and Greeb, 1988; Verma ejb a_l, 1988). According to Carafoli et al (199^), the three functional domains of the pump, namely the P1TC binding domain (Filoteo e_fc al, 1987) that is normally assumed to be part of the binding site for ATP, the domain surrounding the aspartyl phosphate (James et_ al, 1987) and the calmodulin binding domain (Janes et al,' 1988) are located on the cytoplasmic face of the membrane. The calmodulin UNIVERSITY OF IBADAN LIBRARY - 261 - binding domains are made up of positively charged and hydrophobic residues in the N-terminal portion of the domain. In order to assess therefore, the effect of aflatoxin B>| binding to the membrane on the Ca^+- ATPase activity of erythrocyte membrane, the toxin~ was preincubated with the membrane so as to allow for the mobility of the toxin from the carrier solution dimethylsulfoxide, into the membrane. C-â +-ATPase activity was assayed thereafter. The data (Fig. 17) obtained indicated, that the ATPase activity is drastically reduced in the presence of calmodulin. It seems likely that the toxin could interact with calmodulin and thus prevent it from activating the enzyme. The finding that the basal activity of the enzyme was not significantly affected may be explained by the reasoning that aflatoxin could bind to the enzyme only when calmodulin/Ca^+ complex is bound. This fact is supported by the observation that calmodulin stimulated enzyme was inhibited by at least 50a- by the toxin. Although, the possibility UNIVERSITY OF IBADAN LIBRARY 262 - that aflatoxin could bind to the same site as calmodulin or even to calmodulin itself could not be ruled out, it seems possible also that the toxin could bind to the calmodulin-induced conformational state of the enzyme. In this regard, increasing calmodulin concentration has no effect whatsoever on the inhibition of aflatoxin B . This finding demonstrates clearly that the toxin does not bind directly to calmodulin and thus its inhibitory effect is due to its interaction with the pump. The effect of ATP on the extent of inhibition of the enzyme by aflatoxin B-] was studied in order to determine whether the toxin could bind to any of the two ATP binding sites. Several workers have shown that the enzyme has two ATP binding sites, one with low affinity ('14-5-400 uli) the other with high affinity (1-2.5 uM) (Muallem and Karlish, 1979; Richard et al, 1977)* The data obtained in this study (Fig. 19 and 20) demonstrated that the toxin does not significantly affect the affinity of the enzyme for ATP at the low-affinity site with or without calmodulin. Consequently, increasing ATP UNIVERSITY OF IBADAN LIBRARY - 263 - concentration does not reverse the inhibition by aflatoxin. This observation suggests that the toxin does not interfere with the regulation of the enzyme by ATP at the ATF regulatory site. This observation also indicates that the toxin does not bind to the lysine residue (601) responsible for binding ATF at this site. Kinetic analysis of the results (Fig. 20) indicated that only the Vmax of the calmodulin-stimulated enzyme was reduced in the presence of aflatoxin . This finding suggests that the toxin probably affects the turnover rate of the enzyme. The membrane bound enzyme was solubilized in the presence of triton X-100 and eluted from calmodulin-agarose affinity column with a buffer containing, EDTA, phosphatidyl choline and 10% glycerol. The purified enzyme, has a molecular weight ranging between 136-1^0KDa. It is well established that calmodulin has two major effects on the purified Ca^-pumping ATPase; an increase in its maximum velocity and • an enhanced Ca*2' , - UNIVERSITY OF IBADAN LIBRARY 264 - sensitivity, mainly as a result of the conversion of the low Ca -affinity component to a high Ca^+- affinity component (Knauf et_ al, 197̂ -; Gopinath and Vincenzi, 1977)• The effect of the toxin on the purified enzyme (Fig. 21 and 22) further confirmed that, the toxin inhibited the enzyme by about 50%, while it has no effect on the basal activity of the enzyme. It could thus be postulated that irrespective of the state of the enzyme; whether membrane bound or not, the toxin binds to the enzyme protein either 'in situ' or purified. This indicated that the toxin has a specific site to which it binds. Analysis of the influence of the toxin (Fig. 23 and 24) on the kinetic properties of the enzyme showed that the Km and Vmax values of the purif: enzyme are reduced by about 75% and 50%, respectively when compared to the results obtained in the absence of af latoxin B>]. This observation further showed that the toxin could either bind to the calmodulin — binding domain thus preventing the effector from binding, or to another site on the enzyme UNIVERSITY OF IBADAN LIBRARY - 265 - in such a way that the affinity of the enzyme for camodulin is affected. In either case, the stimulation of the enzyme by calmodulin will he reduced in the presence of aflatoxin B^. Calmodulin is a well characterized endogenous calcium-dependent modulator of the calcium pump. (Gopinath and Vincenzi, 1977)- Results of studies by Knauf et al (197^0; Niggli at al (1981) have shown that the catalytic activity of the membrane bound and purified enzyme are enhanced several foldsby calmodulin. These workers deduced that calmodulin alone is not active, except when it binds Ca2£; + . The conformation of the calmodulin p complex becomes more helical and it binds to p the inactive Ca^— ATPase reversiblyJ the inactive enzyme thus becomes an active calcium pump. This Ca + modulator is not the only activator of the enzyme. Ronner et al,(1979) have shown that acidic phospholipids activate the enzyme by increasing p its Ca^ — sensitivity and thus increases its rate of turnover (Niggli et_ al, 1981; Aljobore and Roufogalis, 1981). Although Ca^+ are needed during UNIVERSITY OF IBADAN LIBRARY - 266 - the activation of calmodulin it is not required in the case of phospholipids (Carafoli and Zurini 1982), also the molar ratio of phospholipid to Ca“+ transport ATPase is considerably higher than that for an equivalent activation by calmodulin (Niggli jet al_, 1981), therefore the modes of binding of the two are not the same. Experiments have shown that the number of phospha' tidyl serine molecules required for maximal ATPase activity was near 50 per micelle, implying that a single complete layer of phospholipid molecules surround each of the transmembrane helixes of the ATPase for maximal activity to occur. (Nelson and Hanahan, 1985)• Furthermore, Zvaricth at al_, (1990) were able to show that the amino acid residues between 81 and 76KDa are the phospholipid binding domain. This domain is lysine rich and it consists ofaabout 44 amino acid residues. Several workers have shown that the amount of calmodulin and phospholipids required for stimulation are dramatically different; while calmodulin activates the enzyme at a 1:1 molar UNIVERSITY OF IBADAN LIBRARY - 267 - ratio, optimal activation by phospholipids requires hundred of molecules per molecule of ATPase (Enyedi et al, 1987; Zvaricth et al, 1990; Brodin at al, 1991). Enyedi et al, (1987), have shown that acidic phospholipids are apparently more effective than calmodulin i.e. that they decrease the Km 2( C a + ) to values lower than calmodulin;this has led to the suggestion that acidic phospholipids and calmodulin activate the Ca2^+-ATFase by separate mechanisms involving different binding sites. In order to understand the mechanisms of inhibition of the calmodulin activated enzyme by aflatoxin the purified enzyme was subjected to phospholipid activation in the presence of aflatoxin B^. Results (Fig. 25 ) reported in this thesis showed that the toxin inhibited cardiolipin stimulated enzyme by about 28%. This result indicated that because, phospholipid and calmodulin bind at different sites (Zvaritch et al, 1990), the activation by calmodulin is more significantly UNIVERSITY OF IBADAN LIBRARY 268 - inhibited than that of phospholipid. The result also indicated that the toxin has preference for calmodulin stimulated enzyme. The only reasonable interpretation for the selective inhibition of calmodulin stimulation is that the toxin binds to the enzyme only at a site where further binding to calmodulin is hindered while the toxin does not bind as such to the phospholipid binding site. This interpretation finds support from knowledge on the sequences of the amino acid residues of both the phospholipid and the calmodulin binding domains. Although, aflatoxin B>) has been shown to bind to lysine rich histones, it appears that the lysine-rich phospholipid-domain of the Ca^+- ATPase does not have a significant affinity for the toxin while the calmodulin-binding domain has groups or residues that could bind aflatoxin B^. One possible candidate is tryptophan 1107, The possibility that the toxin could bind to this residues is supported by the finding that tryptophan is the target for aflatoxin B^ binding to UNIVERSITY OF IBADAN LIBRARY - 269 - 3ovine serum albumin (BSA) (Heini and Schabot, 1986). Several studies have shown that detergent alone could not support ATPase activity (Tarverna and Hanahan, 1980) although this depends on the ratio of the detergent to protein used, and on the type of detergent used choice of detergent is also very important since the effect is based on the critical micelle concentration (CMC) of the detergent used. (Taverna and Hanahan, 1989). For instance triton N-100 has a CMC which is about one third that of triton X-100. Triton X-100 is also a very effective detergent for solubilizing the Ca^-ATPase and less is required. The effect of the toxin on the triton X-100 solubilized enzyme Fig. 27 shows that the toxin inhibited by about 50%, the calmodulin triton X-100 activated enzyme when the ratio of triton X-100 to mg protein was about 2, while no effect of toxin was observed at lower triton X-100 to protein ratio. This result indicated that, triton X-100 alone * could not support activation, although it could also UNIVERSITY OF IBADAN LIBRARY - 270 - mean that triton X-100 could possibly be mimicking the effect of phosphatidyl choline when the ratio is 2. The finding that there is no effect at low ratios of triton X-100 to protein shows that the enzyme did not have the right phospholipid or triton X-100 environment required for activity. Apart from calmodulin and phospholipids, limited proteolysis by trypsin and calpain could also activate the enzyme (Wang e_t a_l, 1988). However, studies of limited proteolysis of the purified enzyme have yielded information of great interest on the organization of the functional domains in the molecule. Stieger and Schatmann (1981) have shown that the activation by trypsin corresponds to a decrease of Km (Ca^+) of the pump to levels that under experimental conditions, were even lower than those obtained with calmodulin. However, when it was maximally activated the pump became fragmented into a number of products. Zurini ejt al (1984) have shown that proteolysis for 5-7 mins UNIVERSITY OF IBADAN LIBRARY - 271 - yielded 90KDa fragment that is responsive to both calmodulin and acidic phospholipids. Results reported in this thesis (Table 20) showed that calmodulin stimulated the ATPase octivity of the 90KDa fragment. This activity is inhibited by the toxin. This means that, the site at which the toxin inhibits is somewhere within 90KDa fragment and confirms also that the site of inhibition is the calmodulin binding domain as previously discussed. Further proteolysis of EPTA-treated enzyme showed that the 76KDa product obtained after 90 mins of preincubation was not stimulated by calmodulin and was also not inhibited by the toxin (Table 20). The results indicated that because the calmodulin binding domain has been cleaved in the 76KDa fragment (Wang et al, 1989) the toxin could no 1 longer inhibit the truncated and activated enzyme since the inhibition of the enzyme is at the calmodulin binding domain. This domain has abundance of basic amino acid residues such as- tryptophan and lys*ine residues. (Blumenthal, Tateio, Edelman, Charbonneau, Titatni, Walsh, Krebs, UNIVERSITY OF IBADAN LIBRARY - 2 7 2 - 1983). It seems possible therefore that aflatoxm could be binding to lysine residue, since findings reported by Swenson ejb al (1974) shown that the toxin binds to lysine histone preparations. The toxin may also bind to tryptophan as postulated by Heini and Schabcrt (1986 ) . The mechanism of the binding of the toxin to the calmodulin binding domain may be explained1 from the standpoint of the structure of the calmodulin binding domain (Fig. 4A). Enyedi et_ jal_ (1989) ; Vorherr jrt al_ (1990) have shown that in the absence of calmodulin, the calmodulin binding domain (domain C) binds to domain A and thereby limits assesibility to Ca^+, while in the presence of calmodulin, calmodulin binds to calmodulin- binding domain, and domain A is free to bind Ca^+. However, in the presence of aflatoxin B^, the toxin may bind to domain C thereby preventing the stimulation of the enzyme by calmodulin. To further confirm the binding that the toxin could be binding to the calmodulin binding UNIVERSITY OF IBADAN LIBRARY 273 - domain another proteolytic agent was used. It has been shown that the Ca^ dependent cystein- protease also known as calpain, will cleave about 14KDa fragment from the whole molecule of Ca^+- ATPase, thereby, making the enzyme insensitive to calmodulin. The cleavage removes a part of the domain A required in calmodulin binding. Results of experiments reported in this thesis (Table 19) showed that increasing concentration of aflatoxin B* has no effect on the calpanized ATPase and thus indicated that the possible target of the toxin has been cleaved from the enzyme, since the l24KDa is devoid of a part of the calmodulin binding domain. It is difficult to know which amino acid residue is really involved in aflatoxin binding. However, it could be speculated that both lysine and tryptophan may be possible targets. In order to determine exactly the amino acid residue to which the toxin binds, radio-active labelled aflatoxin will be required. Furthermore, such experi*ments will involve the UNIVERSITY OF IBADAN LIBRARY - 274 - use of High performance liquid chromatography (KPLC) to purify and separate tryptophan fragments to which the toxin is hound. Thus aflatoxin B^- labelled fragment will then he subjected to sequencing. Alternatively, the fragments may he run on an SDS-polyacrylamide gel electrophoresis and the hand transferred by electrohlotting to FVDF/Imobilon F followed by amino acid sequencing of the band corresponding to the aflatoxin- labelled fragment in a sequenator or automatic amino acid sequencer. Several workers have shown that some drugs such as phenothiazine neuroleptics do inhibit calmodulin-stimulated enzyme at low concentrations while at high concentrations of the compounds they also inhibit the basal ATPase activity stimulated by acidic phospholipids and limited proteolysis. Based on these rationalization, it seems possible therefore that the aflatoxin could be mimicking the action of an anticalmodulin drug. In general, it seems possible, therefore that enzymes that have the identical calmodulin binding domain such as Ca£:+ transporting ATFase could be inhibited by UNIVERSITY OF IBADAN LIBRARY - 275 - aflatoxin B^. One major example is cyclic AMP phosphodiesterase which functions in the formation of cAKP. Also, Hidaka, Sasaki, Tanaka, Endo, Ohno, Fujii and Nagata (1981) haveeshown that when anticalmodulin drugs such as phenothiazine, thioxanthene and naphtalene sulfornamide were used to treat CHO cells, these workers deduced that, the growth of these cells were arrested at "both the G/S boundary and in S phase. It seems possible therefore that the toxin could arrest these cells at bothG/S and S phase. This could however be the primary event in the toxicity of this toxin UNIVERSITY OF IBADAN LIBRARY 276 SUMMARY OF RESULTS 1. Incubation of the ghost membrane with aflatoxin B^ shows that the toxin binds sponta­ neously and irreversibly to erythrocyte membrane in a non-co-operative manner. 2. Aflatoxin B^ inhibits the calmodulin stimulated activity of erythrocyte Ca^A+-pumping ATPase while it has no effect on its basal activity^ whereas maximum velocity of the stimulated enzyme is drastically reduced by aflatoxin B^. Also there is no significant effect on the K̂, of the enzyme with or without calmodulin. 3. Aflatoxin B^ inhibits the calmodulin stimulated purified enzyme whereas it has no significant effect on its basal activity. The maximum velocity and the affinity for ATP Km (ATP) of the calmodulin stimulated enzyme were reduced by aflatoxin while in the absence of calmodulin the Km and Vmax *«re not affected by aflatoxin Bi. UNIVERSITY OF IBADAN LIBRARY 277 4. Aflatoxin B-j inhibited the cardiolipin stimulated by about 28%, while it has no signifi­ cant effect on the basal activity of the enzyme. Experiments on the effect of aflatoxin B^ on the stimulation of the enzyme by Triton X-100 shows that Triton X-100 maximally activated the enzyme only when the ratio triton: protein is 2 and aflatoxin inhibited the triton activated enzyme by about 50%. 5. Results from this thesis shows that PC alone did not activate the pump. Calmodulin - stimulated enzyme in the presence of PC was inhibited by aflatoxin B^. In addition,aflatoxin B^ inhibited the PS stimulation of the enzyme by about 28% while the enzyme was not further stimulated by calmodulin. 6. Results of the effect of aflatoxin B^ on proteolysed enzyme shows that aflatoxin B^ has no effect whatsoever on the calpanised enzyme, whereas the toxin inhibited the ATPase activity of the UNIVERSITY OF IBADAN LIBRARY - 278 enzyme partially trypsinised for 5 W. which correspond to the 90KDa fragment. The toxin has no effect on the enzyme trypsinised for 90 which corresponds to the 76KDa fragment of the ATPase. UNIVERSITY OF IBADAN LIBRARY - 279 CONTRIBUTION TO KNOWLEDGE In this thesis, evidence has been presented to show that: 1. Aflatoxin binds to the membrane and can alter the function of the membrane. 2. Aflatoxin B̂ has no effect on the basal activity of the membrane bound and purified ATPase. 3. Aflatoxin B^ inhibits the activity of the calmodulin stimulated membrane bound and purified ATPase. 4. Aflatoxin B̂ inhibits the stimulation of the membrane bound and purified enzyme activity by acidic phospholipids. 5. The Km and Vmax the stimulated enzyme are reduced by aflatoxin 6. Aflatoxin B>| seems to bind at the calmodulin binding domain of the enzyme. UNIVERSITY OF IBADAN LIBRARY 280 - REFERENCES Adamo, H.P., Rega, A.F. and Garrahan, P.J. (1988). Pre-steady-state phosphorylation of the human red cell Ca^+-ATPase. J. Biol. Chem. 2§$: 17548-17554. ____________ (1990). The Eg-- E>| transition of t- he Cap + ATPase from plasma membranes studied by phosphorylation. J. Biol. 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(London) 287: 15-32. UNIVERSITY OF IBADAN LIBRARY 288 Burnside , J.E., Sippel, W.L., Forgacs, J., Caril, W.T., Atwood, M.B. and Doll, E.R. (1957). A disease of swine and cattle caused by eating mouldy corn. II. Experimental production with pure cultures of moulds. Am. J. Vet, Res. 18: 817-824. Cameron, !. (1966). Hyaluronidase and Cancer. New York Pergamon Press, pp. 260-265. Carafoli, E., Tiozzo, R., Lugli, G., Govetti, F. and Kratzing, C. (1974). The release of calcium from heart mitochondria by sodium J. Mol. Cell Cardiol. 6: 361-371. ' ss Carafoli, E. and Crompton, M. (1978). The regula­ tion of intracellular calcium. Curr. Topics. Membr. Transp. 10: 151-216 Carafoli, E. (1981). Calmodulin in the membrane transport of Ca++* Cell Calcium 2: 353-363. ' ss Carafoli, F. (1984). 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