University of Texas at El Paso DigitalCommons@UTEP Open Access Theses & Dissertations 2008-01-01 Effect Of Redox-Cycling Agents On Nitric Oxide Synthase Activity In Vitro And Modulation Of Protein Expression In Vivo: No Nitric Oxide Synthase In Mitochondria Priya Venkatakrishnan University of Texas at El Paso, [email protected] Follow this and additional works at: https://digitalcommons.utep.edu/open_etd Part of the Biology Commons, and the Neuroscience and Neurobiology Commons Recommended Citation Venkatakrishnan, Priya, "Effect Of Redox-Cycling Agents On Nitric Oxide Synthase Activity In Vitro And Modulation Of Protein Expression In Vivo: No Nitric Oxide Synthase In Mitochondria" (2008). Open Access Theses & Dissertations. 376. https://digitalcommons.utep.edu/open_etd/376 This is brought to you for free and open access by DigitalCommons@UTEP. It has been accepted for inclusion in Open Access Theses & Dissertations by an authorized administrator of DigitalCommons@UTEP. For more information, please contact [email protected]. EFFECT OF REDOX-CYCLING AGENTS ON NITRIC OXIDE SYNTHASE ACTIVITY IN VITRO AND MODULATION OF PROTEIN EXPRESSION IN VIVO: NO NITRIC OXIDE SYNTHASE IN MITOCHONDRIA PRIYA VENKATAKRISHNAN Department of Biological Sciences APPROVED: R.T. Miller, Ph.D., Chair Igor C. Almeida, Ph.D. Kristin Gosselink, Ph.D. David Borrok, Ph.D. Patricia D. Witherspoon, Ph.D. Dean of the Graduate School Copyright © by Priya Venkatakrishnan 2008 Dedication To my dearest grandfather Mr. S. Krishna Iyengar, my first guru, a radiantly noble soul and a library of practical wisdom, who continues to teach me the art of living. EFFECT OF REDOX-CYCLING AGENTS ON NITRIC OXIDE SYNTHASE ACTIVITY IN VITRO AND MODULATION OF PROTEIN EXPRESSION IN VIVO: NO NITRIC OXIDE SYNTHASE IN MITOCHONDRIA by PRIYA VENKATAKRISHNAN, B. Pharm (Hons.), M.S. DISSERTATION Presented to the Faculty of the Graduate School of The University of Texas at El Paso in Partial Fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY Department of Biological Sciences THE UNIVERSITY OF TEXAS AT EL PASO December 2008 Acknowledgements First of all, I convey my sincere thanks and gratitude to my mom, Mrs. Renganayaki Venkatakrishnan, who has been with me in all my decisions, sacrificing her interests, supporting morally and financially, whenever needed. I am deeply indebted to my dad, Mr. A. P. Venkatakrishnan for showering me with unconditional love and affection and always believing in my abilities and supporting me in whatever I undertake. I thank my mentor, Dr. R.T. Miller for accepting me as a student at the University of Kentucky as well as at The University of Texas at El Paso, and allowing me to experiment a variety of things by adopting an open-lab system. I am deeply indebted to Dr. Miller for trusting my abilities and results, allowing me to pursue my interests in the laboratory, teaching me how to analyze data and communicate with researchers in a scientific manner. Above all, I owe all my scientific writing abilities to Dr. Miller as he has been patiently helping and correcting me all throughout my doctoral degree. I thank my Ph.D advisory committee members, Dr. Igor C. Almeida, Dr. Kristin Gosselink, Dr. David Borrok for their valuable suggestions and guidance throughout my Ph.D. Special thanks to Dr. Almeida for all the valuable inputs that he gave during the proteomic analyses. Also, I thank Dr. Gary Gairola and my friend, Ernesto S. Nakayasu for their valuable advice and suggestions, but for which I would not have been able to complete my projects. I thank my ex-labmates, Josh for teaching me how to do the biochemical experiments and, Javier for helping me get acclimated in the laboratory, and my current lab-mates, Dr. Joshi and Berenise for being very supportive and taking care of the lab management thereby, allowing me to finish Ph.D at ease. Also, I would like to thank all my friends for being extremely supportive, listening and helping me to stay focused. Special thanks to Harish, Maria, Kalyan and Sujan for their guidance and the last- v minute support while writing the dissertation. I also would like to thank Dr. Miller for providing my stipend from his grant # ES 011982 from the National Institute of Environmental Health Sciences (NIEHS)/NIH. Also, this project was also supported in part by grant (# 5G12RR008124) to the Border Biomedical Research Center (BBRC)/University of Texas at El Paso from the National Center for Research Resources (NCRR)/NIH and by grant (# 03-RDE- 005) from the Kentucky Science and Education Foundation (KSEF). vi Abstract This dissertation describes studies conducted in three areas dealing with the interaction of redox-active compounds with the flavin- and heme- containing enzyme, neuronal nitric oxide synthase (NOS). They are as follows: I. Studies were initiated using extracts from several varieties of tobacco to identify constituent(s) that could interact with nNOS. Results from [14C]-L-arginine to [14C]-L-citrulline conversion assays to which tobacco extracts were individually added, indicate that there are nNOS inhibitors present in tobacco and they are non-polar, lipophilic and non-volatile in nature. In addition, results from assays in which the tobacco-derived redox-active compound, 2,3,6- trimethyl-1,4-naphthoquinone or 2-methyl-1,4-naphthoquinone were individually incorporated into the nNOS activity assays indicate that single compounds can produce either stimulation or inhibition of L-citrulline production depending on the concentration of the quinone to which nNOS is exposed DVZHOODVWKHFRPSRXQG¶VHOHFWURFKHPLFDOFKDUDFWHULVWLFV II. Data, both for and against the presence of a mitochondrial NOS isoform (mtNOS) is in the refereed literature. However, irrefutable evidence, either for or against the existence of a mtNOS isoform has not been forthcoming. Therefore, studies were initiated at our laboratory to investigate whether any NOS isoform resides in ultra pure rat liver mitochondria, utilizing 3 independent techniques. Results from activity assays, mass spectrometry (MS) analyses and immunochemical detection for NOS, independently, as well as collectively, refute the claim that a NOS isoform exists within rat liver mitochondria, at least down to the limit of detection. vii III. Mitochondria are thought to be involved in modulating the toxicity of redox-active compounds. In order to understand the effect of 1, 1-dimethyl-4,4-bipyridinium ion (paraquat), a redox-active compound, on levels of protein expression in whole liver homogenate as well as rat liver mitochondria during the early stage of paraquat exposure (40 mg/kg, i.p), proteomic analyses were performed 3 hrs after paraquat administration. Mass spectrometric analyses of paraquat-treated rats identified 37 proteins in rat liver mitochondria as well as in whole rat liver homogenate that were either down- and/or up-regulated, at least 2 folds above control levels. GO ontology and Interpro scan analyses identified that the modulated proteins in rat liver mitochondria were oxidoreductases and transferases. In whole rat liver homogenate, the modulated proteins were mainly oxidoreductases, transferases as well as proteins involved in protein binding functions. Additionally, rat liver proteins involved in ubiquitination and the proteolytic pathway were modulated. In addition to these studies, a cost-effective method was designed and validated for obtaining and purifying the chicken antibodies against the stable, reactive nitrogen species biomarker, 3-nitrotyrosine. viii Table of Contents Acknowledgements..........................................................................................................................v Abstract......................................................................................................................................... vii List of Tables ................................................................................................................................ xii List of Figures.............................................................................................................................. xiii List of Illustrations.........................................................................................................................xv Introduction......................................................................................................................................1 References.............................................................................................................................17 Chapter 1: Naphthoquinones and Bioactive Compounds from Tobacco as Modulators of Neuronal Nitric Oxide Synthase Activity.............................................................................31 1.1 Introduction...........................................................................................................................31 1.2 Experimental Procedures ......................................................................................................35 1.3 Results and Discussion .........................................................................................................39 1.4 Conclusion ............................................................................................................................58 1.5 Acknowledgements...............................................................................................................59 1.6 References.............................................................................................................................60