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Information to Users INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandardm a r g ins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. University Microfilms International A Bell & Howell Information Company 300 North Zeeb Road. Ann Arbor. Ml 48106-1346 USA 313/761-4700 800/521-0600 Order Number 91S0S67 Studies on the metabolism of oxalate, glyoxylate, glycolate and glycine by peroxisomes and mitochondria from rat liver Sutherland, Steven Thomas, Ph.D. The Ohio State University, 1991 UMI 300 N. Zeeb Rd. Ann Arbor, MI 48106 STUDIES ON THE METABOLISM OF OXALATE, GLYOXYLATE, GLYCOLATE AND GLYCINE BY PEROXISOMES AND MITOCHONDRIA FROM RAT LIVER DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Steven Thomas Sutherland, B.S. ***** The Ohio State University 1991 Dissertation Committee: Approved by K.E. Richardson j / / c> (P * / /? G.P. Brierley Adviser Department of Medical A.J. Merola Biochemistry •To Mom and Dad ii ACKNOWLEDGEMENTS I wish to express sincere appreciation to Dr. Keith E. Richardson for his guidance and patience throughout the research. Thanks go to the members of my advisory committee, Drs. Gerald P. Brierley and A. John Merola. Special thanks go to Dr. Richard H. Nuenke for his assistance. VITA September 14, 1956 .... Born - Takoma Park, Maryland 1978 .................... B.S., Andrews University Berrien Springs, Michigan 1979-1980 .............. Graduate Teaching Associate, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio 1982-1985 .............. Assistant Chemist and Associate Chemist, Loma Linda Foods, Mt. Vernon, Ohio 1986-1988 .............. Graduate Research Associate, Department of Physiological Chemistry, The Ohio State University, Columbus, Ohio 1988-1990 .............. Biochemistry Tutor, College of Dentistry, The Ohio State University, Columbus, Ohio FIELDS OF STUDY Major Field: Medical Biochemistry iv TABLE OF CONTENTS DEDICATION .............................................ii ACKNOWLEDGEMENTS ................................... iii VITA .................................................iv LIST OF TABLES vi LIST OF FIGURES .................................... viii LIST OF ABBREVIATIONS ................................ xi INTRODUCTION ....................................... 1 CHAPTER I LITERATURE REVIEW ...................... 3 CHAPTER II MATERIALS AND METHODS .................. 52 CHAPTER III RESULTS AND DISCUSSION ................ 71 CONCLUSIONS ..........................................125 APPENDIX A: DATA FOR THE METABOLISM OF GLYOXYLATE AND GLYCOLATE IN RAT LIVER MITOCHONDRIA AND PEROXISOMES ..................................... 130 APPENDIX B: DATA FOR THE METABOLISM OF GLYOXYLATE AND GLYCOLATE IN RAT LIVER MITOCHONDRIA AND PEROXISOMES—II ................................. 134 APPENDIX C: DATA FOR THE EFFECTS OF ALANINE, GLUTAMATE AND ORNITHINE ....................... 138 APPENDIX D: DATA FOR THE EFFECTS OF DL-PHENYL- LACTATE ......................................... 142 LIST OF REFERENCES ................................. 129 V LIST OF TABLES 1. Purity of [l-,4C]glyoxylate. Identity of contaminants....................................... 73 2. Specific activities of marker enzymes ........... 75 3. Peroxisomal uptake of oxalate, glyoxylate, glycolate and glycine. Radioactivity retained with peroxisomes.................................. 83 4. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Metabolic products ............................. 102 5. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-ll. Metabolic products ............................. 106 6. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Metabolic products ............................. Ill 7. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Metabolic products......................................... 116 8. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of the initial incubations ............ 131 9. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column separations . 132 10. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions .......... 133 vi 11. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of the initial incubations ............ 135 12. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of the AG 50W-X8 column separations . 136 13. Metabolism of glycolate, glyoxylate and glycine in rat liver mitochondria and peroxisomes-II. Results of glycolate, glyoxylate and oxalate determinations of formate fractions .......... 137 14. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the initial incubations....................... 139 15. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column s e p a r a t i o n s ............. 140 16. Effects of alanine, glutamate and ornithine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions .......... 141 17. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the initial incubations....................... 143 18. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of the AG 50W-X8 column separations............. 144 19. Effects of DL-phenyllactate and alanine on the metabolism of glycolate and glyoxylate in rat liver mitochondria and peroxisomes. Results of glycolate, glyoxylate and oxalate determinations of formate fractions .......... 145 vii LIST OF FIGURES 1. Precursors of urinary oxalate ................... 9 2. Structures of oxalate, glyoxylate and glycolate . 10 3. Pathways of oxalate biosynthesis ............... 11 4. Peroxisomal, mitochondrial and cytosolic compartmentalization of glyoxylate metabolism. 16 5. Pathway of oxalate biosynthesis from ascorbic acid in m a n ...................................... 25 6. Pathway for the biosynthesis of oxalate from ascorbic acid in animals other than man and other primates.................................... 28 7. Pathways of oxalate biosynthesis from various carbohydrates ................................... 33 8. Pathways for the conversion of xylitol to oxalate 3 6 9. Metabolic lesions of primary hyperoxaluria type I and type I I .................................... 42 10. Pathway for the metabolism of ethylene glycol . 48 11. Modified Lowry protein determination standard c u r v e ............................................ 59 12. Phosphorus standard curve ....................... 65 13. 14C quench curve for the Beckman LS 7000 liquid scintillation counter ........................... 70 14. Chromatogram of [1-I4C] glyoxylate using a radioactive flow detector ....................... 72 viii 15. Swelling of rat liver mitochondria in iso-osmotic solutions of ammonium glycine, ammonium glyoxylate and ammonium glycolate ........................... 78 16. Mechanism of glyoxylate, glycolate and glycine uptake by mitochondria ........................... 79 17. Swelling of rat liver mitochondria in iso-osmotic solutions of ammonium oxalate ................... 80 18. Mechanism of oxalate uptake by mitochondria . 81 19. Uptake of oxalate by peroxisomes.................. 84 20. Uptake of glyoxylate by peroxisomes ............. 85 21. Uptake of glycolate by peroxisomes .............. 86 22. Uptake of glycine by peroxisomes.................. 87 23. The enzymatic reactions in glyoxylate metabolism occurring in mammalian systems .................. 89 24.
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