PHOSPHOLIPID DEPENDENCY OF MEMBRANE-ASSOCIATED PYRIDINE NUCLEOTIDE-UTILIZING AND SUCCINATE DEHYDROGENASE ACTIVITIES OF ADULT HYMENOLEPIS DIMINUTA (CESTODA) AND ASCARIS SUUM (NEMATODA) Carl Breidenbach A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 2012 Committee Carmen Fioravanti, Advisor Jill Zeilstra-Ryalls Raymond Larsen © 2012 Carl Breidenbach All Rights Reserved iii ABSTRACT Carmen Fioravanti, Advisor The adult intestinal cestode, Hymenolepis diminuta, is essentially anaerobic in its metabolism and generates ATP without the need for oxygen. H. diminuta relies upon a mitochondrial NADPH→NAD transhydrogenase to link the NADPH produced by the pyruvate- forming arm of the malate dismutation reaction, catalyzed by the mitochondrial “malic enzyme”, with the NADH-requiring, anaerobic electron transport system. The electron transport-coupled fumarate reductase serves to reduce fumarate, the terminal electron acceptor, to succinate. A phospholipid dependency was established previously with respect to the transhydrogenase, fumarate reductase, and the lesser NADH oxidase. Of the phospholipids assessed, the transhydrogenase exhibited a phosphatidylcholine preference. The present study expands on prior findings by using phospholipase A1, A2, C and D, organic solvent, and ammonium sulfate treatments of H. diminuta mitochondrial membranes. Other reduced pyridine nucleotide-utilizing systems viz., NAD(P)H cytochrome c reductase, NADH→NAD transhydrogenation, NAD(P)H-, and lipoamide dehydrogenase activities as well as succinate dehydrogenase were evaluated. A comparative study also was undertaken by treatment with the phospholipases of isolated mitochondrial membranes from the anaerobic intestinal nematode, Ascaris suum. The data presented indicate a phospholipid dependence not only of the previously reported systems, but of membrane-associated mitochondrial systems in H. diminuta and A. suum. H. diminuta NADH cytochrome c reductase displayed phospholipid dependence based on phospholipase A2 and C treatments, and a neutral lipid dependence based on organic solvent iv treatments. Ammonium sulfate fractionation had little effect. Succinate dehydrogenase activity displayed phospholipid dependence based on phospholipase C and organic solvent treatments. Ammonium sulfate fractionation decreased succinate dehydrogenase activity, but phosphatidylcholine supplementation further diminished activity. A. suum NADH cytochrome c reductase, NADH oxidase and fumarate reductase systems exhibited phospholipid dependence based on phospholipase A2 and C treatments. Interestingly, A. suum succinate dehydrogenase appeared more resistant to phospholipase treatment than the corresponding H. diminuta system. v ACKNOWLEDGMENTS I would like to thank Dr. Carmen Fioravanti for his help and support while researching and writing this thesis. Also, special thanks for Elizabeth Shuler for helping me start my work in the laboratory and her continued aid while doing my thesis research. I would also like to thank my family, David, Sandra, Kurt and Dirk Breidenbach as well as my girlfriend Brittany Murphy, for their support, especially during the writing process of this thesis. I would like to thank the Department of Biological Sciences at Bowling Green State University for funding my research and a special thanks to Sigma Xi, The Scientific Research Society for their Grant in Aid of Research. vi TABLE OF CONTENTS Page CHAPTER I. LITERATURE REVIEW ............................................................................... Hymenolepis diminuta .............................................................................................. 1 Ascaris suum ............................................................................................................ 4 Phospholipid Dependence ........................................................................................... 7 Purpose ............................................................................................................ 9 CHAPTER II. THE STUDY INTRODUCTION …………………………………………………………………….12 MATERIALS AND METHODS ............................................................................... 14 RESULTS Treatment of H. diminuta mitochondrial membranes with phospholipases .. 19 Extraction of H. diminuta mitochondrial membranes with organic solvents 21 Effect of phospholipid addition to partially lipid-depleted H. diminuta mitochondrial membrane preparations .............................................. 21 Treatment of Ascaris suum mitochondrial membranes with phospholipases 36 DISCUSSION ............................................................................................................ 47 LITERATURE CITED .......................................................................................................... 55 APPENDIX I: INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) Approval Information ............................................................................................... 59 vii LIST OF TABLES Table Page 1 Effect of solvent extraction on reduced pyridine nucleotide-utilizing and succinate dehydrogenase activities of adult Hymenolepis diminuta mitochondrial membranes 34 2 Effect of 30-55 ammonium sulfate fractionation and addition of phosphatidylcholine on reduced pyridine nucleotide-utilizing and succinate dehydrogenase activities in adult Hymenolepis diminuta mitochondrial membranes 35 3 Titration of phospholipase A2 on Hymenolepis diminuta mitochondrial membrane- associated NADPH→NAD transhydrogenase ........................................................... 53 viii LIST OF FIGURES Figure Page 1 Life cycle of Hymenolepis diminuta .......................................................................... 2 2 Pathway of primary carbohydrate utilization in Hymenolepis diminuta ................... 3 3 Life Cycle of Ascaris suum ........................................................................................ 6 4 Cleavage of phospholipids by phospholipases .......................................................... 10 5 Effects of phospholipases on percent differences in NADH cytochrome c reductase activity of adult Hymenolepis diminuta mitochondrial membranes .......................... 24 6 Effects of phospholipases on percent differences in NADPH cytochrome c reductase activity of adult Hymenolepis diminuta mitochondrial membranes .......................... 25 7 Effects of phospholipases on percent differences in NADH→NAD transhydrogenation activity of adult Hymenolepis diminuta mitochondrial membranes .......................... 26 8 Effects of phospholipases on percent differences in NADH dehydrogenase activity of adult Hymenolepis diminuta mitochondrial membranes............................................ 27 9 Effects of phospholipases on percent differences in NADPH dehydrogenase activity of adult Hymenolepis diminuta mitochondrial membranes............................................ 28 10 Effects of phospholipases on percent differences in succinate dehydrogenase activity of adult Hymenolepis diminuta mitochondrial membranes............................................ 29 11 Effects of phospholipases on percent differences in NADH oxidase activity of adult Hymenolepis diminuta mitochondrial membranes..................................................... 30 12 Effects of phospholipases on percent differences in lipoamide dehydrogenase activity of adult Hymenolepis diminuta mitochondrial membranes............................................ 31 ix 13 Effects of phospholipases on percent differences in fumarate reductase activity of adult Hymenolepis diminuta mitochondrial membranes..................................................... 32 14 Effects of phospholipases on percent differences in NADPH→NAD transhydrogenase activity of adult Hymenolepis diminuta mitochondrial membranes .......................... 33 15 Effects of phospholipases on percent differences in NADH cytochrome c reductase activity of adult Ascaris suum muscle mitochondrial membranes ............................. 38 16 Effects of phospholipases on percent differences in NADPH cytochrome c reductase activity of adult Ascaris suum muscle mitochondrial membranes ............................. 39 17 Effects of phospholipases on percent differences in NADH→NAD transhydrogenation activity of adult Ascaris suum muscle mitochondrial membranes ............................. 40 18 Effects of phospholipases on percent differences in NADH dehydrogenase activity of adult Ascaris suum muscle mitochondrial membranes .............................................. 41 19 Effects of phospholipases on percent differences in NADPH dehydrogenase activity of adult Ascaris suum muscle mitochondrial membranes .............................................. 42 20 Effects of phospholipases on percent differences in succinate dehydrogenase activity of adult Ascaris suum muscle mitochondrial membranes .............................................. 43 21 Effects of phospholipases on percent differences in NADH oxidase activity of adult Ascaris suum muscle mitochondrial membranes ....................................................... 44 22 Effects of phospholipases on percent differences in lipoamide dehydrogenase activity of adult Ascaris suum muscle mitochondrial membranes .............................................. 45 23 Effects of phospholipases on percent differences in fumarate reductase activity of adult