Downloaded for Personal Non-Commercial Research Or Study, Without Prior Permission Or Charge
Total Page：16
File Type：pdf, Size：1020Kb
https://theses.gla.ac.uk/ Theses Digitisation: https://www.gla.ac.uk/myglasgow/research/enlighten/theses/digitisation/ This is a digitised version of the original print thesis. Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten: Theses https://theses.gla.ac.uk/ [email protected] Studies on the Energy Metabolism of Herpetomonads by Christopher A. Redman Department of Zoology University of Glasgow Thesis presented in submission for the degree of Doctor of Philosophy in the Faculty of Science. September 1991. ProQuest N um ber: 11011434 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 11011434 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 ACKNOWLEDGEMENTS I would like to thank Professor R. S. Phillips for the use of the facilities of the Department of Zoology, Doctor Barbara Lockwood for help and advice on the use of HPLC and FPLC, Doctor Colin Robertson for advice on the electrophoresis, and David Laughland for help with cell cultures. I acknowledge the Science and Engineering Research Council for the funding for this work. I also acknowledge Doctor Laurance Tetley for allowing me to use his electron micrographs in the introduction to this thesis. I would also like to thank Isobel Thompson, Patricia Johnston and Liz Denton for their many kindnesses during my stay in the Zoology Department. I wish to thank Professor G. H. Coombs for helpful discussions and guidance during the course of my Research. Finally, I dedicate this thesis to my parents, without whom I would never have come this far. ABBREVIATIONS ADP adenosine diphosphate ALDO aldolase AMP adenosine monophosphate ATCC American type culture collection ATP adenosine triphosphate ATPase adenosine triphosphatase BzPFR benzoyl-prolyl-phenylanalyl-aranyl-p- nitro-analide cAMP cyclic adenosine monophosphate C 0 2 carbon dioxide CoA coenzyme A CTP cytidine triphosphate Da Daltons DHAP dihydroxyacetone phosphate DNA deoxyribonucleic acid DTT dithiothreitol f flagellum FAD+(H2) flavine adenine dinucleotide (reduced) F-1,6 -P2 frucose- 1,6 -bisphosphate F-2.6-P2 fructose-2, 6 -bisphosphate FR fumarate reductase GAPDH glyceraldehyde-3-phosphate dehydrogenase GAPDHC cytosolic GAPDH GAPDHg glycosomal GAPDH GDP guanosine diphosphate GK glycerol kinase GlcNH 2 glucosamine GlcNAc N-acetyl glucosamine GMP guanosine monophosphate G-3-P glycerol-3-phosphate GPDH glycerol-3-phosphate dehydrogenase GPI glycosylphosphatidyl inositol GIPL glycoinositol phospholipid GPI-PLC GPI-specific phospholipase C GTP guanosine triphosphate HBSS Hank’s balanced salts solution HCO3' hydrogen carbonate HDL high density lipoprotein Hepes N-[2-hydroxyethyl]piperazine-N’-[2- ethanesulphonic acid] HIFCS heat inactivated foetal calf serum HK hexokinase IMP inosine monophosphate k kinetoplast LDL low density lipoprotein LIT liver infusion tryptose LPG lipophosphoglycan m microbody Man mannose MDH malate dehydrogenase ME ‘malic’ enzyme mRNA messenger ribonucleic acid mt mitochondrion n nucleus NAD+(H) nicotinamide adenine dinucleotide (reduced) NAD-ICDH NAD+-specific isocitrate dehydrogenase NADP(H) nicotinamide adenine dinucleotide (reduced) NADP-ICDH NADP-specific isocitrate dehdrogenase 0 2 oxygen PAGE polyacrylamide gel electrophoresis PC pyruvate decarboxylase PEP phosphoenolpyruvate PEPCK phosphoenolpyruvate carboxykinase PFK phosphofructokinase 3-PGA 3-phosphoglyceric acid PGI phosphoglucoseisomerase PGK phosphoglycerate kinase PGKC cytosolic PGK PGKg glycosomal PGK Pj inorganic orthophosphate PI phosphatidylinositol PI-PLC phosphatidylinositol-specific phospholipase C PK pyruvate kinase PSP promastigote surface protease Q quinone RER rough endoplasmic reticulum RNA ribonucleic acid SCFA short-chain fatty acid SDH succinate dehydrogenase SDS sodium dodecyl sulphate SHAM salicylhydroxamic acid TCA tricarboxylic acid cycle TEA triethanolamine TEMED N.N.N’.N’-tetramethylethylenediamine TIM triosephosphate isomerase Tris Tris(hydroxymethyl)aminomethane TS thymidylate synthase UDP uridine diphosphate UMP uridine monophosphate UrPRTase uracil phosphoribosyltransferase UTP uridine triphosphate VSG variant surface glycoprotein CONTENTS Page TITLE i ACKNOWLEDGEMENTS ii ABBREVIATIONS iii CONTENTS vii LIST OF FIGURES xii LIST OF TABLES xiv SUMMARY xv 1.0 INTRODUCTION 1 1.1 The Trypanosomatidae 2 1.1.1 The taxonomy of Herpetomonas 2 1.1.2 Life cycle of Herpetomonas 6 1.2 Ultrastructure of the Trypanosomatidae 8 1.3 Trypanosomatid metabolism 20 1.3.1 Anabolic processes 21 1.3.1.1 Nucleic acids 21 1.3.1.1.1 Purine metabolism 22 1.3.1.1.2 Pyrimidine metabolism 24 1.3.1.1.3 Replication and transcription 25 1.3.1.2 Proteins 27 1.3.1.3 Lipids 30 1.3.1.3.1 Lipid anchors 31 1.3.2 Catabolic processes 38 1.3.2.1 Substrate utilization and end products 38 1.3.2.2 Effects of gaseous conditions 41 1.3.2.3 Catabolic pathways 44 1.3.2.3.1 Glycolysis and the pentose phosphate shunt and their regulation 44 1.3.2.3.2 Carbon dioxide fixation pathways 47 1.3.2.3.3 The tricarboxylic acid cycle 50 1.3.2.3.4 The respiratory chain 55 1.3.2.3.5 Proteinases 60 1.3.3 Glycosomes 64 Aims of the project 71 MATERIALS AND METHODS 72 Cells 73 2.1.1 Maintenance and cultivation 73 2.1.2 Harvesting of the cells 73 Enzyme analysis 74 2.2.1 Preparation of cell homogenates for enzyme assays 74 2.2.2 Enzyme assays 74 2.2.3 Limits of the enzyme assays 79 Protein determinations 79 Metabolite analysis 80 2.4.1 Long-term incubations 80 2.4.2 Short-term incubations 81 2.4.2.1 Harvesting of cells 81 2.4.2.2 Incubations conditions 81 2.4.3 Analysis using enzyme-based assays 82 2.4.3.1 Preparation of extracts 82 2.4.3.2 Enzyme-bases assays 83 2.4.4 High Performance Liquid Chromatography (HPLC) 84 2.4.4.1 Preparation of extracts 84 2.4.4.2 Analysis using the Polypore H column 85 2.5 Studies on the subcellular location of the enzymes 85 2.5.1 Particulate and soluble activities 85 2.5.2 Fractionation by differential centrifugation 86 2.5.2.1 Cell lysis 86 2.5.2.2 Differential centrifugation 86 2.6 Proteinases 87 2.6.1 pH profile of total cellular proteinase activity 87 2.6.2 Gelatin gel analysis of extracellular activities 87 2.6 .2.1 Gelatin-SDS-PAGE preparation 87 2.6.2.2 Proteinase staining 88 2.6.2.3 pH profile of extracxellular activities 88 2.7 Purification and characterisation of pyruvate kinase of H. ingenoplastls 89 2.7.1 Modifying the pyruvate kinase assay 89 2.7.2 Solubilization of the particulate enzymes 89 2.7.3 Enzyme purification 89 2.7.3 .1 Cell lysis and preparation 90 2.7.3.2 Anion exchange chromatography 90 2.7.3.3 Gel filtration chromatography 91 2.7.4 Polyacrylamide gel electrophoresis 91 2.7.4.1 Staining for protein 91 2.7.5 pH profile of pyruvate kinase activity 92 2.7.6 Enzyme kinetics 92 2.8 M aterials 92 3.0 RESULTS 94 3.1 Glucose catabolism 95 3.1.1 Glycolysis and the pentose phosphate shunt 95 3.1.2 Glucose consumption 95 3.1.3 Cell growth and glucose catabolism 99 3.1.4 Short-term incubations 99 3.1.5 HPLC analysis 106 3.1.6 Organic acid production 111 3.1.7 Enzyme pathways of catabolism 122 3.2 Proteinases 128 3.2.1 Cellular proteinase actvity 128 3.2.2 Extracellular proteinase activity 128 3.3 Subcellular distribution of enzymes 136 3.4.1 Purification of PK from H. ingenoplastis 140 3.4.2 Analysis of PK purified from H. ingenoplastis 152 3.4.2.1 pH profile 152 3.4.2.2 The effects of divalent cations 152 3.4.2.3 The effects of KCI 152 3.4.2.4 The effects of ADP 166 3.4.2.5 The effects of PEP 166 3.4.2.6 Inhibition by ATP 166 3.4.2.7 Activation by f '2’6_p2 167 3.4.2.8 Other effectors of PK 167 4.0 DISCUSSION 168 4.1 Glucose catabolism in Herpetomonads 169 4.1.1 Enzymes of glycolysis and the pentose-phosphate shunt 169 4.1.2 Analysis of metabolite consumed and produced <1 jq 4.1.3 Pathways of glucose catabolism 183 4.1.4 Proteinases 187 4.1.5 Subcellular organisation of metabolism 188 4.16 The pyruvate kinase of H. ingenoplastis 192 4.2 The anaerobic nature of H. ingenoplastis 194 5.0 REFERENCES 199 LIST OF FIGURES Page 5 10 11 12 29 30 31 32 52 53 54 100 103 109 113 117 119 129 131 133 142 Xm 22 144 23 146 24 148 25 152 26 154 27 156 28 158 29 160 30 162 31 164 Scheme A 172 Scheme B 174 LIST OF TABLES Page 1 4 2 6 3 96 4 97 5 98 6 107 7 108 8 112 9 121 10 123 11 123 12 124 13 126 14 127 15 136 16 137 17 138 18 150 19 166 SUMMARY Herpetomonas ingenoplastis is an intriguing and unusual trypanosomatid with many anaerobic features, including the lack of a complete cytochrome chain, the presence of an acristate mitochondrion and the ability to grow equally well under aerobic and anaerobic conditions.