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The Ionophore Nigericin Transports Pb2+ with High Activity and Selectivity: a Comparison to Monensin and Ionomycin

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LEAD TRANSPORT PROPERTIES OF CARBOXYLIC ACID AND SYNTHETIC IONOPHORES DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of the Ohio State University By Shawn A. Hamidinia, B.A. ***** The Ohio State University 2005 Dissertation Committee: Approved by Professor Douglas Pfeiffer, Adviser Professor Ruth Altschuld _________________________ Adviser Professor Ross Dalbey Biophysics Program Professor Thomas Clanton ABSTRACT Metal ion transport studies were performed using naturally occurring carboxylic acid ionophores, a synthetic ionophore, and phospholipid vesicles. Several compounds were identified that are highly selective for the transport of Pb2+ compared to physiological monovalent and divalent cations. Based upon their respective second-order rate constants, the compounds nigericin and monensin were shown to be selective for the transport of Pb2+, with nigericin being slightly more selective in that regard. Plots of log rate vs. log Pb2+ or log ionophore concentration, in addition to pH dependency, indicate that the predominant transporting species are NigPbOH or MonPbOH. Agents that collapse membrane potential were not required to achieve a high transport rate, which is indicative of an electroneutral mechanism. Nigericin and monensin catalyzed transport of Pb2+ are only modestly affected by physiological concentrations of Ca2+, Mg2+, Na+, or K+. These findings led to the testing of monensin as a therapeutic agent for Pb intoxication using an intact rat model. Monensin at a concentration of 100 ppm in the feed was given to rats exposed to 100 ppm Pb(acetate)2 in drinking water over a three week period. A reduced Pb ii accumulation in several organs and tissues was shown. Furthermore, there was an acceleration in the excretion of Pb without depleting organs of essential trace metals such as Zn2+ and Cu2+. An additional study showed that the co-administration of 100 ppm monensin with meso-dimercaptosuccinate after an exposure to 100 ppm Pb(acetate)2 in drinking water for three weeks significantly reduced the Pb content from femur, brain, and heart without markedly perturbing the concentrations of physiological elements. Thus, monensin may be useful for the treatment of Pb poisoning when combined with DMSA. KTC-15-cr-5 was synthesized and found to be an effective ionophore for Pb2+ and Cd2+ transport across a phospholipid bilayer. The results suggest that Pb2+ and Cd2+ are primarily transported as a 1:1 complex and by an electrogenic mechanism. The high selectivity for Pb and Cd is of possible value for Pb/Cd intoxication and furthermore to wastewater treatment. iii Dedicated to my family iv ACKNOWLEDGMENTS I wish to thank my adviser, Douglas Pfeiffer, for his guidance, encouragement, and support which made this thesis possible. My deep gratitude goes to Warren Erdahl for intellectual support and encouragement. I also wish to thank those in the lab including, Gregory Steinbaugh, Clifford Chapman, Jittendra Kumar, and Ron Louters who have provided me with consistent support and a very friendly work environment. This research was supported by grant GM 66206 from the National Institute of General Medical Sciences, National Institutes of Health, by grant 0255017B fom the American Heart Association, Ohio Valley Affiliate, and by Grant HR00-030 from the Oklahoma Center for the Advancement of Science and Technology. v VITA December 15, 1975..................Born - Bridgeport, Connecticut 1998..........................................B.A. Biochemistry, Case Western Reserve University 1998-present.............................Graduate Teaching and Research Associate, The Ohio State University PUBLICATIONS Research Publication 1. Hamidinia, S.A., Tan, B., Erdahl, W.L., Chapman, C., Taylor, R.W., and Pfeiffer, D.R. The ionophore nigericin transports Pb2+ with high activity and selectivity: A comparison to monensin and ionomycin. Biochemistry. 43, 15956- 15965, 2004. 2. Hamidinia, S. A., Shimelis, O. I., Tan, B., Erdahl, W. L., Chapman, C. J., Renkes, G. D., Taylor, R. W., and Pfeiffer, D. R. Monensin mediates a rapid and selective transport of Pb2+. The Journal of Biological Chemistry. 277(41): 38111- 38120. 2002. 3. Wang, E., Erdahl, W. L, Hamidinia, S. A., Chapman, C. J., Taylor, R. W., and Pfeiffer, D. R. Transport properties of the calcium ionophore ETH-129. Biophysical Journal. 81, 3275-3284, 2001. vi FIELDS OF STUDY Major Field: Biophysics vii TABLE OF CONTENTS Page Abstract…...................................................................................................……………. ii Dedication...................................................................................................……………. iv Acknowledgments.......................................................................................……………. v Vita............................................................................................................…………….. vi List of Tables.............................................................................................…....……….. xi List of Figures..............................................................................................………..…. xii List of Abbreviations..................................................................................………….... xv Chapters: 1. Introduction and Background...............................................................…..…………... 1 1.1 Properties of ionophores………………………………………….………….1 1.2 Model membrane system…………………………………………….…….... 3 1.3 The impact of Pb toxicity……..……………………………………...……... 7 1.4 Current treatment for Pb intoxication………….........…………….………… 8 1.5 The discovery that ionomycin is a Pb2+ ionophore………………..…………9 2. Monensin mediates a rapid and selective transport of Pb2+: Possible application of monensin for the treatment of Pb2+ intoxication….………………………….………11 2.1 Introduction..........................................................................………………..11 2.2 Materials and Methods.................................................................…………..14 Reagents and solvents………………………………..………………… 14 Preparation of phospholipid vesicles……………………….………….. 15 Pb2+ buffers and the determination of transport………………….……..17 Potentiometric titrations and the determination of pH in aqueous methanol……………………………………………………… 19 viii Treatment of experimental animals……………………..…………...… 21 Determination of Pb in biological samples….……………………….…21 2.3 Results…………………………………………………….……………….. 23 Monensin mediated Pb2+ transport………………...…………...……… 23 Competitive relationships between Pb2+ and other cations……………. 25 Monensin promotes the excretion of Pb2+ in rats..……..…………….... 27 2.4 Discussion………………………………………………………………….. 29 2.5 Summary…………………………………………………..………….……. 34 3. Monensin improves the effectiveness of meso-dimercaptosuccinate when used in the treatment of Pb intoxication……….……………….………….…………... 52 3.1 Introduction………………………………………………………………....52 3.2 Materials and methods………………………………………….………….. 54 Treatment of experimental animals...……………...…………...……… 54 The determination of Pb and other elements by ICP-MS….…...……… 56 3.3 Results……………………………………………………………………....57 Levels of Pb and other elements following a period of Pb administration………………………………………………………..… 57 Depletion of previously accumulated Pb……………….….…...……… 59 Effects of monensin alone on the levels of other elements...…...……... 60 Pertubations produced by monensin in DMSA treated rats..…...………61 Pertubations produced by monensin plus DMSA compared to no treatment……………………………………………….....……… 62 Effects of treatment on other parameters………………......…...……… 63 3.4 Discussion………………………………………………………………….. 63 3.5 Summary…………………………………………………………………… 70 4. The ionophore nigericin transports Pb2+ with high activity and selectivity: A comparison to monensin and ionomycin…………………………...……………….. 91 4.1 Introduction......................................................................…………………. 91 4.2 Materials and methods…..................................................…………………. 94 Reagents and solvents……………………………………...…...……… 94 Potentiometric titrations and the determination of pH in aqueous methanol.…...………………………………………………… 94 4.3 Results……………………………………………………….……………...95 4.4 Discussion…………………………………………………………..…….. 100 4.5 Summary………………………………………..……….……………....... 104 5. Selective transport of Pb2+ and Cd2+ by a Kemp’s triacid capped 15-crown-5 ether across a phospholipid bilayer membrane…………………………….……….....116 5.1 Introduction………………………………………………….………….....116 ix 5.2 Materials and Methods... ……………………………….….…………….. 117 Reagents…………………………………………………...…..………117 Synthesis of crown ether derivative………….…………...…...………118 Determination of transport………………………………...…......……118 Pb2+ and Cd2+ buffers……………………………………...…...……...119 5.3 Results……………………………………………………………………..119 5.4 Discussion………………………………………………………………… 122 5.5 Summary…………………………………………………….……………. 126 6. Discussion…………………………………………………………………………. 136 6.1 Conclusion.....................................................................................……….. 136 6.2 Future Perspectives......................................................…………………… 139 Literature Cited..........................................................................................….………... 142 x LIST OF TABLES Table Page 2.1 Monensin effects on Pb2+ accumulation…………………...….……….. 49 2.2 Monensin effects on Pb2+ clearance…………………………………… 50 2.3 Monensin effects on trace cation levels……………………………….. 51 3.1 Effects of Pb administration on the levels of selected elements in
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