Factors affecting the 16α-hydroxylation of estrone 3-sulfate by guinea pig liver microsomes

Abstract

The estrone 3-sulfate 16α-hydroxylase of guinea pig liver microsomes has been demonstrated to be sensitive to CO. A CO/O2 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 Mg2+ or Ca2+ while the same concentration of Mn2+ resulted in a twofold increase. Lesser increases were seen with Na+ or K+ and complete inhibition was obtained in the presence of Fe2+, Cu2+, 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. The nonionic detergent, Brij 35, caused least inhibition of all and, when hepatic microsomes were treated with higher concentrations of Brij 35, about 80% of protein and over 95% cytochrome P450 were to be found in the 100 000 × g supernatant. Microsomal activity was more stable when stored at −20 °C in buffer containing glycerol, EDTA, and dithiothreitol than in buffer alone. Under best conditions only 10% of the hydroxylase activity was lost in one week.