Cytochrome P-450 for 11β- and 18-hydroxylase activities of bovine adrenocortical mitochondria: one enzyme or two?

Abstract

The 11.beta.- and 18-hydroxylase activities of a highly purified cytochrome P-450 from bovine adrenocortical mitochondria were examined in detail with a view to determining whether the 2 activities are shown by a single protein or by 2 distinct proteins. The purified enzyme shows a single N-terminal residue (glutamic acid) and its amino acid composition is reported. Both enzyme activities decay considerably during storage at 4.degree. C for 11 days and the rates of decay are similar for the 2 activities. Metyrapone inhibits both activities competitively (Ki = 1.50 and 1.43 .mu.M for 11.beta.- and 18-hydroxylase, respectively). CO inhibits both activities and the ratio CO:O2 for 50% inhibition is similar for the 2 activities (K = 1.69 and 1.53). A variety of nonspecific inhibitors produce approximately the same inhibition of both activities. Antiserum produced by rabbits to the purified enzyme on double diffusion in agarose gels gives a single band with the purified enzyme. Increasing concentrations of antiserum added to the assay system produce increasing and proportionate inhibition of both activities. The evidence strongly supports earlier suggestions that the 2 hydroxylase activities occur in a single protein.