Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions |
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Authors: | D R Dutton S K McMillen A J Sonderfan P E Thomas A Parkinson |
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Institution: | 1. Department of Soil System Sciences, Helmholtz Centre for Environmental Research –UFZ, Halle (Saale), Germany;2. Soil Science, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany;1. Division of Psychological Medicine and Clinical Neurosciences, MRC Centre for Neuropsychiatric Genetics and Genomics, Cardiff University, Wales, United Kingdom;2. Cwm Taf Morgannwg University Health Board, Wales, United Kingdom;3. MRC Integrative Epidemiology Unit, University of Bristol, Bristol, United Kingdom;4. Nic Waals Institute, Lovisenberg Diaconal Hospital, Oslo, Norway;5. Population Health Sciences, Bristol Medical School, University of Bristol, Bristol, United Kingdom;6. School of Psychology, Cardiff University, Wales, United Kingdom;1. Yanchi Research Station, School of Soil and Water Conservation, Beijing Forestry University, Beijing, 100083, China;2. Beijing Engineering Research Center of Soil and Water Conservation, Beijing Forestry University, Beijing, China;3. Beijing Vocational College of Agriculture, Beijing, 102442, China;4. Faculty of Forestry and Environmental Management, 28 Dineen Drive, University of New Brunswick, Fredericton, New Brunswick, E3B 5A3, Canada |
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Abstract: | The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone. |
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