Download (8Mb)

A Thesis Submitted for the Degree of PhD at the University of Warwick Permanent WRAP URL: http://wrap.warwick.ac.uk/136759 Copyright and reuse: This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. For more information, please contact the WRAP Team at: [email protected] warwick.ac.uk/lib-publications STUDIES OF MITOCHONDRIA!. TRANSFORT SYSTEMS A Thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy at the University of Warwick, Department of Molecular Sciences By AZIZ HAMOUD HASAN AL-LAM I, B.Sc.,M .Sc. May, 1977 I TABLE OF CONTENTS Table of Contents -L-ê? List of Tables ^ List of Figures iy. Abbreviations viii Acknowledgements x Summary xi CHAPTER 1 INTRODUCTION 1.1 Ceneral introduction 1 1.2 Architecture of mitochondria and localization of their functions •) 1.3 Proposed mechanisms for oxidative phosphorylation 5 1.4 Inhibitors and uncouplers of oxidative phosphorylation used in the present study 6 1.5 Ion carrier systems 11 1 .6 The coupling of metabolite transport to electron transport 24 1.7 Bindings of Inhibitors to mitochondria and submitochondrial particles 26 32 1.8 ATP- Pi exchange reaction 26 1.9 The aim of this thesis 27 CHAPTER 2 ADP TRANSLOCA3 ION IN MITOCHONDRIA 2. 1 Introduction 28 2.2 Materials and methods 29 2.3 Results 33 2.4 Discussion 58 TABLE OF CONTENTS Page Table of Contents i List of Tables iii List of Figures iv Abbreviations viii Acknowledgements X Summary x l CHAPTER 1 INTRODUCTION 1.1 Ceneral introduction 1 1.2 Architecture of mitochondria and localization of their functions 1 1.3 Proposed mechanisms for oxidative phosphorylation 5 1.4 Inhibitors and uncouplers of oxidative phosphorylation used in the present study 6 1.5 Ion carrier systems 11 1.6 The coupling of metabolite transport to electron transport 24 1.7 Bindings of inhibitors to mitochondria and submitochoudrial particles 26 32 1.8 ATP- Pi exchange reaction 26 1.9 The aim of this thesis 27 CHAPTER 2 ADP TRANSLOCATION IN MITOCHONDRIA 2. 1 Introduction 28 2.2 Materials and methods 29 2.3 Results 33 2. 4 Discussion 58 U Page CHAPTER 3 3 14 H-ATRACTYLOSIDE. C -HEXACHLOROPHENE AND RHODAMINE 6G BINDING TO MITOCHONDRIA 3.1 Introduction 61 3.2 Materials and Methods 63 3.3 Results 65 3.4 Discussion 87 CHAPTER 4 CALCIUM TRANSPORT IN MITOCHONDRIA 4.1 Introduction 91 4.2 Materials and Methods 93 4.3 Results 96 4.4 Discussion 114 CHAPTER 5 THE ATP- 2Pi EXCHANGE REACTION 5.1 Introduction 116 5.2 Materials and Methods 117 5.3 Results 119 5.4 Discussion 129 CHAPTER 6 THE MECHANISM OF PHOSPHATE AND DICARBOXYLATE TRANSPORT IN MITOCHONDRIA 6.1 Introduction 131 6.2 Materials and Methods 133 6.3 Results 135 6.4 Discussion 151 CHAPTER 7 GENERAL DISCUSSION AND CONCLUSIONS 154 REFERENCES 157 t iii LIST OF TABLES TABLES Page 1.1 Calcium-dependent reactions in cells 21 2.1 Effect of inhibitors, uncouplers and ionophores on ADP uptake 40 2.2 Effect of rhodamine compounds on ADP uptake 42 2.3 Influence of incubation period on iniiibition of ADP uptake by agaric acid, sulfobromophthalein and hexachlorophene 43 3.1 Effects of ADP, inhibitors and anaesthetics on 14c-HCP binding to mitochondria 74 14 3.2 Effect of uncouplers on C -HCP binding to mitochondria 75 3.3 Effect of incubation period and temperature on 1 -HCP binding to mitochondria 75 3.4 Comparison between the bidning of rhodamine compounds to mitochondria 77 3.5 Effect of incubation periods, temperature and pH on Rh6G binding to mitochondria 84 4. 1 Release of mitochondrial calcium by inhibitors, uncouplers and ionophores 103 32 5. 1 Inhibition of ATP- Pi exchange reaction in submitochondrial particles 127 32 6.1 Inhibition of phosphate (' P i) transport into mitochondria 143 \ . X iv LIST OF FIGURES FIGURES Page 1.1 Schematic representation of the mitochondria 2 1.2 Schematic representation of inner mitochondrial membrane as made up of tripartite repeating units 2 1.3 Schematic representation of coupling scheme in conformational model of oxidative phosphorylation 2 1.4 Scheme of mitochondrial structure and compartmentation 4 1.5 Structures of atractyloside (A tr), sulfobromophthalein (SBP)( agaric acid (A A \ rhodamine 6G (Rh6G )( rhodamine B (RhB) and bongkrekic acid (BKA ) 7 1.6 The metabolite flux through the mitochondrial membrane 12 1.7 ADP carrier models 14 — 2 — 1.8 Sequential coupling of phosphate > ADP and ATP4" transport to electrochemical gradient generated by electron transport 17 1.9 The role of the ADP> ATP carrier in mitochondrial phosphate transfer reactions 19 1.10 Uptake of calcium and phosphate 22 1.11 Important transport systems of the inner membrane in rat-liver mitochondria 25 2.1 Tim e course of the uptake of ADP 34 14 2.2 Tim e course of the C-ADP efflux in exchange with external ADP 35 2.3 Double-reciprocal plot of the exchange of ADP 37 14 2.4 Effect of Atr on the kinetics of C-ADP efflux in exchange with external ADP 38 2.5 Inhibition of ADP translocase by SBPt agaric acid and HCP 45 2.6 Double-reciprocal plot of the exchange of ADP in the presence of SBP and agaric acid 46 V FIGURES Page 2.7 Double-reciprocal plot of the exchange of ADP in the presence of HCP 47 2. 8 Inhibition of the ADP uptake by agaric acid and SBP; presented in the form of a Dixon plot 48 2.9 The effect of pH on the inhibition by BKA( HCP SBP and agaric acid of ADP uptake 50 2.10 Influence of pH on inhibition of ADP uptake by BKA 51 2.11 Effect of Atr( SBP and AA on ^C-ADP binding 53 2.12 Inhibition of specific ADP binding by SBP and AA 54 2.13 Effect of agaric acid, SBP, HCP and Atr on the DNP-stimulated ATPase activity 56 2.14 Effect of agaric acid, Atr, SBP and HCP on submitochondrial particles ATPase 57 3 3.1 Effect of ADP on ' H-atractyloside binding to mitochondria 66 3 3. 2 Effect of SBP on H-atractyloside binding to mitochondria 67 3 3.3 Effect of agaric acid on H-atractyloside binding to mitochondria 68 3 3.4 Effect of HCP on H-atractyloside binding to mitochondria 69 3 3.5 Inhibition of H -atractyloside binding to submitochondrial particles 71 14 3.6 C-HCP binding to mitochondria 72 14 3.7 C-HCP binding to submitochondrial particles 73 14 3.8 Relationship between C-HCP binding and phospholipid composition of mitochondria 76 14 3.9 Effect of washing on C-HCP binding to mitochondria 78 3.10 Effect of leucinostatin and calcium on Rh6G binding to mitochondria 80 3.11 Effect of TE T and DBCT on Rh6G binding to mitochondria 81 3.12 Effect of valinomycin and 1799 on Rh6G binding to mitochondria 82 vi FIGURE Page 3.13 Effect of butacaine and amytal on Rh6G binding to mitochondria 83 3.14 Relationshio between Rh6G binding and phospho lipid composition of mitochondria 86 3.15 Effect of washing on Rh6G binding to mitochondria 88 4.1 Inhibition of calcium stimulated respiration in rat liver mitochondria by HCP 92 | | 4. 2 Energy-linked Ca transport in mitochondria 93 4.3 Effect of pH on calcium uptake 97 4.4 Rh6G inhibition of ATP stimulation of calcium accumulation 98 4. 5 Effect of ruthenium red, DNP and HCP on calcium uptake by mitochondria 99 45 -H- 4.6 Inhibition of Ca uptake by mitochondria 100 4.7 Effect of DBCT and TE T on calcium uptake 102 | | 4.8 Maintenance of accumulated Ca in mitochondria 105 45 ++ 4.9 Effect of ruthenium red and HCP on "Ca binding 110 4.10 The effect of DUCT, TE T and DNP on 45Ca++ binding 111 j j 4.11 Relationship between Ca binding and phospho lipid composition of mitochondria 113 32 5. 1 Rate of ATP- Pi exchange reaction 120 32 5. 2 Effect of pH on ATP- Pi exchange reaction 120 5.3 Effect of ATP concentrations on exchange reaction 121 32 32 5.4 Effect of ' 'Pi concentration on ATP- Pi exchange reaction 122 32 5.5 Effect of Atr> SBP> AA and Rh6G on ATP- Pi exchange reaction 123 32 5.6 Effect of HCP, DCCD and TET on ATP- Pi exchange reaction 124 32 5.7 Effect of oligomycin and venturicidin on ATP- Pi exchange reaction 125 FIGURE Page 32 5.8 Effect if FCCP, 1799, PCP and DNP on ATP- PL exchange reaction 128 6.1 Scheme of sequential action of metabolite carriers 131 32 6.2 Time dependence of Pi exchange from medium to mitochondria 136 6.3 Lineweaver-Burk plot of dependence of rates of 32pi exchange on medium phosphate concentration 137 32 6.4 Effect of SBP, p-CMBA on Pi uptake 139 32 6.5 Effect of mersalyl and ionophores on Pi uptake 140 32 6.6 Effect of HCP and FCCP on Pi uptake 141 6.7 infect of SBP, mersalyl and p -CMBA on the Pi -Pi exchange 145 14 6.8 Kinetic analysis of the inhibition of C-malate uptake by SBP, using the double reciprocal plot 146 14 6.9 Kinetic analysis of the inhibition of C-succinate uptake by SBP, using :- A, Dixon plot, and B, double reciprocal plot 147 1 4 6.10 Effect of SBP on the kinetics of C-malate efflux in exchange with external malate 148 14 6.11 Effect of SBP on the kinetics of C-malate efflux in exchange with external Pi 149 14 6.12 Effect of SBP on the kinetics of C-succinate efflux in exchange with external succinate 150 v iii ABBREVIATIONS AA Agaric acid ADP Adenosine-5-diphosphate Amytal 5-Ethyl-5-isoamylbarbituric acid ATP Adenosine-5-triphosphate ATPase Adenosine-5 -triphosphatase Atr Atractyloside A 20668 Leucinostatin BKA Bongkrekic acid BSA Bovine serum albumin CAT Carboxyatractyloside Cl-CCP m -Chlorocarbonyl cyanide phenylhydrazone CPDS 6, 6-dithiodinicotinic acid DBCT Dibutylchloromethyltin chloride DCCD N( N-Dicylcohexyl-carbodiimide DNA Deoxyribonucleic acid DNP Dinitrophenol DTT Dithiothreitol EDTA Ethylenediamine-tetra-acetic acid EGTA Ethyieneglycol-bis-(S-aminoethyl ether)N^N- tetraacetic acid Soluble mitochondrial ATPase F 1 FCCP p -Trifluoromethoxy -carbonyl cyanide -phenyl hydrazone g gram(s ) HCP Hexachlorophene Hepes N-2-hydroxyethylpiperazine N-2-ethanesulfonic acid K Michaelis constant rn L Litre m metre M Molar (concentration) min minute (time) ix V- micro (10 ^ x) NADH Reduced nicotinamide adenine dinucleotide , -9 .

Load more