A 450K arsenite-binding protein of the rabbit liver cytosol Item Type text; Dissertation-Reproduction (electronic) Authors Bogdan, Gregory Michael 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 23/09/2021 18:28:12 Link to Item http://hdl.handle.net/10150/186945 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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Ann Arhor, MI4R 106 A 450K ARSENITE-BINDING PROTEIN OF THE RABBIT LIVER CYTOSOL by Gregory Michael Bogdan 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 Gregory Michael Bogdan entitled A 450K ARSENITE-BINDING PROTEIN OF THE RABBIT LIVER CYTOSOL and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy Date I I / //~~ ~,/, 'I Daniel C.(Liebler /{(CLU\, -~2~~1~( Date ((!2S!y({ Thomas Date Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the dissertation to xhe 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. Di!e!ti~ H. Vasken Aposhinn 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 rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment 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 judgment the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained by the author. 4 ACKNOWLEDGMENTS I am deeply indebted to many people who have come into my life during the course of this work. Many have expanded my knowledge by relating their own experiences, some have worked alongside me sharing their talents, several have advised me in overcoming research obstacles and numerous others have just been there to provide friendship and support. The individual who has helped me the most and been there through it all is Dr. H. Vasken Aposhian. His support, understanding, commitment and friendship were always unwavering. I wish to extend my gratitude to the members of my committee, Drs. Daniel C. Liebler, A. Jay Gandolfi, Klaus Brendel and Tom Lindell, for their time and counsel. They always managed to keep me focused and directed for which I am truly thankful. I would like to express my gratitude to Wallace Clark for his work in protein sequencing and the time he spent advising me on the subject. I would like to thank the individuals who have been apart of the Aposhian laboratory over the years, Dr. Rich Maiorino, Mary Aposhian, David Bruce, Jon Akins, Wei Zheng, Dr. Katie Hurlbut, Debbie Levine, Yuan Wu, Dr. ZhoufaXu, Dr. Robert Zakaharian, Lydia Simon and, especially, Russell Ingersoll. They have all made my experiences truly rewarding and helped in ways too numerous to list. I would like to thank Adriana Sampayo-Reyes and Dr. Narsa Goud for their help with this research project while at this laboratory. This research was supported in part by the Superfund Basic Research Program NIEHS Grant #ES- 04940. Many outside the laboratory have played an enormous role in helping me during this research. Their friendship and love have been instrumental in making my seven years here in Tucson the most memorable part of my life. I would especially like to thank Todd Kennedy, John Sherry, Frank Flomerfelt, Pam Murray, Scott Bailey, Tara Bailey and Brian Bateman. These individuals have greatly affected my life and their friendship will always be cherished. I wish to thank my family for giving me all the opportunities I could ever hope for and being so supportive of all my endeavors. Finally, I wish to thank Stephen Rivera for all the patience, understanding, support and love he has given me over the years. He came into my life during this project and encouraged me throughout its completion. I don't know what the future holds but I do know that with him by my side I could face anything. 5 TABLE OF CONTENTS LIST OF FIGURES ........................................................................................................ 6 LIST OF TABLES ........................................................................................................... 8 ABSTRACT ................................................................................................................... 9 CHAPTER 1. INTRODUCTION ........................................................................................................ 11 Arsenic: Poison and Medicinal Agent.. ......................................................... 11 Arsenic Exposure and Toxicity ...................................................................... 16 Arsenic Chelating Agents ............................................................................... 25 Animal and Human Metabolism Studies ..................................................... 27 Methylation Mechanisms ................................................................................ 40 The Marmoset Anomaly ................................................................................. 46 CHAPTER 2. A MODEL SYSTEM FOR STUDYING CYTOSOLIC PROTEINS WHICH BIND ARSENIC ............................................................................................ 48 Experimental Procedures ................................................................................ 50 Results ................................................................................................................ 56 Discussion ......................................................................................................... 69 CHAPTER 3. INITIAL PURIFICATION OF SPECIFIC CYTOSOLIC PROTEINS WHICH BIND ARSENITE ............................................. 73 Experimental Procedures ................................................................................ 74 Results ................................................................................................................ 79 Discussion ......................................................................................................... 99 CHAPTER 4. FURTHER PURIFICATION OF THE 450K ARSENITE-BINDING PROTEIN ..................................................................... 103 Experimental Procedures ................................................................................ 105 Results ................................................................................................................ 113 Discussion ......................................................................................................... 138 CHAPTERS. CONCLUSIONS ........................................................................................................... 143 REFERENCES ............................................................................................................... 150 6 LIST OF FIGURES Figure Page 1.1 Structures of three arsenic chelating agents............................................. 13 1.2 Structures of two