Date of Award


Degree Type


Degree Name

Doctor of Philosophy


Hydroxylation reactions play an important role in steroid metabolism. The enzyme systems responsible for most steroid hydroxylations are believed to be mixed function oxidases usually acting with reduced pyridine nucleotides as electron donors and cytochrome P450 as the terminal oxidase. A reliable procedure for the assay of live microsomal 16(alpha)-hydroxylation of estrone-3-sulfate has been developed for the guinea pig. It is based on the rapid, quantitative separation of estradiol and estriol by Sephadex LH-20 columns after the chemical reduction and hydrolysis of the incubation products.;Microsomal preparations and incubation conditions that optimized the 16(alpha)-hydroxylation of estrone-3-sulfate were employed. Under these circumstances, reduction of the substrate at C-17 and hydrolysis of the sulfate were minimized. The hydroxylation reaction had an absolute requirement for NADPH, which could not be satisfied by NADH. Apparent Km values for estrone-3-sulfate and NADPH, under the conditions used, were 14 (mu)M and 0.17 mM respectively. 16(alpha)-Hydroxylase assays were conducted under conditions which yielded rates that were zero order with respect to NADPH and estrone-3-sulfate concentration and were linear with respect to incubation time and enzyme concentration.;The estrone-3-sulfate 16(alpha)-hydroxylase of guinea pig liver microsomes has been demonstrated to be sensitive to CO. A CO/O(,2) ratio of 0.64 caused 50% inhibition of activity. Since inhibition was also obtained in the presence of 2-diethylaminoethyl-2,2-diphenylvalerate HCl it seems likely that the hydroxylase is a cytochrome P450 containing system.;A fourfold increase in enzyme activity was brought about by 40 mM Mg('2+) or Ca('2+) while the same concentration of Mn('2+) resulted in a two-fold increase. Lesser increases were seen with Na('+) or K('+) and complete inhibition was obtained in the presence of Fe('2+), Cu('2+), or EDTA.;When assayed in the presence of detergent concentrations sufficiently small to guard against cytochrome P450 destruction, it was found that Cutscum, Triton X-100, and Triton N-101 each caused greatest inhibition of enzyme activity. Lesser inhibition was apparent in the presence of Miranol H2M, cholate, or deoxycholate. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of school.) UMI



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