INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly fi'om the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter face, while others may be fi'om 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, substandard margins, 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 comer and continuing fi’om 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. UMI A Beil & Howell Information Company 300 North Zed) Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 THE SUBCELLULAR DISTRIBUTION OF RAT LIVER AND YEAST GLUCOSE-6-PHOSPHATE DEHYDROGENASE AND ITS ROLE IN REGULATION OF THE ENZYME DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Karen Elizabeth Reilly, B.S. ***** The Ohio State University 1997 Dissertation Committee; Approved by John B. Allred, Advisor Sylvia A. McCune Advisor Karla L. Roehrig Graduate Program in Food Science and Nutrition UMI Number: 9813339 UMI Microform 9813339 Copyright 1998, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 ABSTRACT GIucose-6-phosphate dehydrogenase (G6PD) belongs to the group of lipogenic enzymes that all react similarly under lipogenic conditions. Specifically, when fasted rats are refed a high carbohydrate diet, enzyme activity may increase up to ten times the fasted level in 72 hours. Extensive research has been done to explain this phenomenon for G6PD, and the general conclusion is that the overshoot is caused by increased protein synthesis. However, the possibility that there is activation of the enzyme has not been ruled out. Recent work has shown that other lipogenic enzymes are glycoproteins and may have GPI anchors that contribute to their regulation by shifting the enzymes from particulate to soluble fractions. G6PD was analyzed to see if it too shared these characteristics. The sensitive digoxigenin-labeling method was used to confirm that G6PD is a glycoprotein. SDS-PAGE analysis showed matching migration patterns for protein, activity, antibody reactivity, and carbohydrate staining for both yeast and rat liver G6PD. Treatment of yeast mitochondria with phosphatidyl inositol specific phospholipase c resulted in increased enzyme in the supernatant indicating that G6PD was bound to the mitochondria via a GPI anchor. Additionally, digitonin fractionation of rat liver mitochondria showed that G6PD is associated with the outer mitochondrial membrane. ii Male, Sprague Dawley rats were fed a high carbohydrate diet or fasted for 48 hours and refed a high carbohydrate diet and G6PD activity and quantity measured in the cytosol and mitochondria to determine the subcellular distribution of the enzyme. G6PD activity in the mitochondrial-free supernatant was 2.5 to 3.5 times greater in refed rats compared to fed rats. In the mitochondria, the activity was up to twice as high in refed rats. There was no difference in G6PD quantity between fed and refed rats in either cellular compartment. Mitochondria had ten times as much enzyme as the mitochondrial-free supernatant, but the activity was only one tenth as much. When expressed as actual enzyme specific activity (mU/amount enzyme), the mitochondrial-free supernatant G6PD was seven times higher in refed rats compared to fed rats. The refed rats had twice as much mitochondrial specific activity. Results showed that mitochondrial G6PD is very low in activity while the mitochondrial-free supernatant became much more active when fasted rats were refed a high carbohydrate diet. These results indicated that the overshoot in G6PD activity was due to increased activation of the enzyme which may be unrelated to the presence of carbohydrate attached to the protein or shifts in subcellular location. Kinetic studies were performed with G6PD in fed and refed rats to explain better the increased activation of the enzyme. The Vmax for fed rats was 0.041mU/mg protein versus 0.096mU/mg protein for refed rats. These results were expected, however an unexpected difference in Km values was found. Fed rats had a Km of 28.8uM while refed rats had a Km of 55.9uM. These results show that there may be a different form of the enzyme present in refed rats that has distinct characteristics. Ill Dedicated to my husband, John, and to the little one who provided a deadline for me to complete this project. IV ACKNOWLEDGMENTS There are many people to acknowledge who contributed to my graduate experience. Foremost is my advisor. Dr. John Allred, who is a wonderful mentor and has become a good friend. I realize that the advisor-student relationship is typically not perfect, but I think in this case there was a good mesh of personalities which resulted in a friendly, companionable work environment. Dr. Allred is a great role model for an educator and researcher and some day I hope to be half as successful with my students as he is with his. I thank him for his guidance and patience, and seemingly photographic memory, but most of all his companionship. I would like to thank Drs. Roehrig and McCune for taking the time to be on my committee and for their manuscript suggestions. I also appreciate having them as educators who fostered creative thinking and problem solving. And I thank them for their willingness to lend an ear or provide a piece of advice when the situation warranted. I thank Richard Jurin for the same ability. Thank you to all my fellow students who became fnends and made my time at Ohio State enjoyable—Jason Chou, Luther Chuang, Dave Smith, Heidi Senokozliefif, Isabel Schuermeyer, and Kristin Pape. I truly don't think I could have survived without you. I'm grateful for the brainstorming sessions, one-on-one advice, and most of all the camaraderie. There's more to life than research and studies and we discovered that together. The best times were the times I could wander down the hail and find a fiiendly ear to provide a little break for a while. Thank you all for that. Finally, I would like to thank my husband, John. Your support over the past four and a half years has been astounding. From the first stress-filled quarters when going back to school was a foreign concept to the past few months when the final crush was on, you were always there to boost my spirits and pick up the slack. I know you say you're proud of me for what I've accomplished, but I'm just as proud of you for going through this with me. VI VITA November 22, 1968.......................... Bom - Akron, OH 1991................................................... B.S. Chemistry, John Carroll University 1991 - 1993...................................... Research Scientist New Projects Department The Lubrizol Corporation WicklifFe, OH 1993 - 1994...................................... Graduate Research Assistant The Ohio State University 1994 - Present................................... USDA National Needs Fellow The Ohio State University PUBLICATIONS Research Publications 1. Reilly KE, Allred JB. (1995) Glucose-6-Phosphate Dehydrogenase from Saccharomyces cerevisae is a glycoprotein. Biochem. and Biophys. Res. Com. 216(3):993-8. 2. Allred JB, Reilly KE. Short-term regulation of acetyl CoA carboxylase in tissues of higher animals. (1996) Progress in Lipid Research, Elsevier Press, Oxford 35(4):371-85. FIELDS OF STUDY Major Field; Food Science and Nutrition Vll TABLE OF CONTENTS Abstract.........................................................................................................ii Dedication .....................................................................................................iv Acknowledgments ......................................................................................... v Vita.............................................................................................................. vii List of Tables................................................................................................ x List of Figures.............................................................................................. xi List of Abbreviations ................................................................................. xiii Chapters: Introduction ....................................................................................................1 1. Literature Review ....................................................................................4 GIucose-6-Phosphate Dehydrogenase .................................................4