Functional characterization of hepatic microsomal and heterologousely expressed rabbit cytochrome P450 2B enzymes Item Type text; Dissertation-Reproduction (electronic) Authors Grimm, Scott Wayne. Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 06/10/2021 12:38:56 Link to Item http://hdl.handle.net/10150/186952 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. 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Contact UMI directly to order. UMJ[ A Bell & Howell Informallon Company 300 North Zeeb Road. Ann Arbor. M148106·1346 USA 313.'761·4700 800:521·0600 Order Number 9517564 Functional characterization of hepatic microsomal and heterologous ely expressed rabbit cytochrome P450 2B enzymes Grimm, Scott Wayne, Ph.D. The University of Arizona, 1994 U·M·K 300 N. Zeeb Rd. Ann Arbor, MI 48106 FUNCTIONAL CHARACTERIZATION OF HEPATIC MICROSOMAL AND HETEROLOGOUSLY EXPRESSED RABBIT CYTOCHROME P450 2B ENZYMES by Scott Wayne Grimm A Dissertation Submitted to the Faculty of the COMMITTEE ON PHARMACOLOGY AND TOXICOLOGY (GRADUATE) In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 1994 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Final Examination Committee, we certify that we have read the dissertation prepared bY~S~c~o~t~t~W~ayYun~e~G~r_jwmwm~ _____________________ entitled FUNCTIONAl CHARACTERIZATION OF HEPATIC MICROSOMAl AND HETEROLOGOUSLY EXPRESSED RABBITT CYTOCHROME P4S0 2B EXZYMES and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy ----------------~~~-------------------- Date l II f/ /4'1 Date /I~//c;-~ Date ; I/,/:IAI/ Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. JIV)veJ) )2, ) I e"'1)u>b' Date James R. Halpert 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at The University of Arizona and is deposited in the University Library to be made available to borrowers under the rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College when in his or her judgement the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. 4 ACKNOWLEDGMENTS My sincere gratitude is due to Dr. James Halpert for willingly taking me under his direction as my research advisor, for challenging me, encouraging me, and mentoring me. I would also like to thank the other members of my advisory committee, Dr. Martin Dyroff, Dr. Daniel Liebler, Dr. Hugh E. Laird II, and Dr. Josephine Lai for encouraging me and helping me keep a straight course in the somewhat unorthodox situation of performing much of my research 2300 miles away from the University of Arizona. I thank ZENECA Pharmaceuticals for financial support for my family during my leave of absence to the University of Arizona in 1991. A special thanks is due to Dr. Martin Dyroff and Dr. Chris Rhodes at ZENECA Pharmaceuticals for supporting my ambitions to return to graduate school, for winning the approval of the upper management at ZENECA to pursue this goal, and generously giving me time to perform some of my research during work hours. I gratefully acknowledge the time spent by Dr. Karen Kedzie and Dr. Jeff Stevens to teach me new techniques in molecular biology and cytochrome P450 purification and analysis. I also acknowledge Dr. Richard Philpot and Dr. John Bend for supplying me with some of the materials to conduct this research and for their helpful advice during these investigations and during the preparation of the manuscripts for publication. I sincerely appreciate the prayers, encouragement, and support of my parents, family, and friends during my stressed and tired moments. 5 DEDICATION This dissertation is dedicated to my wife and children. To Kathy, who supported me with her love and patience and unselfishly dedicated the last 4Y2 years of her life to raising our two young sons, Evan and Jeremy. I also dedicate this to Evan and Jeremy, who unknowingly gave up much of our precious time together during their early years. 6 TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................... 9 LIST OF TABLES ........................................................................................................... 10 ABSTRACT .................................................................................................................... 11 1. INTRODUCTION ...................................................................................................... 12 Xenobiotic Metabolism ............................................................................................ 12 The Cytochrome P450 Dependent Monooxygenase System ................................... 13 The Molecular Biology of Cytochromes P450 ........................................................ 14 Cytochrome P450 Nomenclature ..................................................................... 15 Molecular Aspects of Cytochrome P450 Regulation and Function ................. 16 Structure-Function Relationships of Cytochromes P450 ......................................... 17 Characterizing Substrate Specificities of Cytochromes P450 .................................. 19 Heterologous Expression of Cytochromes P450 ............................................ 20 Cytochrome P450 Inhibition ............................................................................ 21 Mechanisms of Cytochrome P450 Inhibition .......................................... 21 Inhibitors as Tools to Study the Function of Individual Cytochrome P450 Forms .............................................................................................. 23 Cytochrome P450 Induction .................................................................................... 24 Polymorphic Expression of Cytochrome P450 Genes ............................................. 26 Microheterogeneity in the Cytochrome P450 2B Subfamily ................................... 28 Cytochromes P450 2B in Rats ......................................................................... 29 Cytochromes P450 2B in Rabbits .................................................................... 30 Research Problem Addressed in This Dissertion ..................................................... 33 Significance and Implications .................................................................................. 33 Organization of Dissertation .................................................................................... 35 2. FUNCTIONAL COMPARISONS OF PURIFIED AND HETEROLOGOUSL Y EXPRESSED RABBIT CYTOCHROMES P450 2B4, 2B-BX, 2B-B 1, AND 2B5 ......................................................................................... 36 Background .............................................................................................................. 36 Materials and Methods ......................... " .................................................................. 38 Materials .......................................................................................................... 38 Animals, Treatments, and Microsome Preparation ......................................... 39 Strains and Media .......... , ................................................................................. 39 COS-7 Cell Expression Plasmids ..................................................................