Purification of a soluble rat liver protein that stimulates microsomal 4-methyl sterol oxidase activity.

A soluble rat liver protein that stimulates microsomal methyl sterol oxidase activity has been isolated and purified to homogeneity by salt fractionation, differential heat inactivation, two calcium phosphate gel association, and Sephadex filtration. The protein, as isolated, may be dissociated with detergent into subunits with a molecular weight of approximately 10,300, as determined electrophoretically. In addition to this stimulatory protein, postmicrosomal supernatant fraction of rat liver contains a low molecular weight methyl sterol oxidase inhibitor, possibly cholesterol ester, which is removed during protein purification. Purified soluble protein enhances the observed rate of oxidative attack of the methyl sterol substrates, 4, 4-dimethyl-5alpha-cholest-7-en-3beta-ol and 4alpha-methyl-5alpha-cholest-7-en-3beta-ol. Addition of increasing amounts of either partially purified or homogeneous soluble protein yields hyperbolic stimulation, from which a K'm of about 7 muM has been calculated (based on a monomeric molecular weight of 10,300). Sequential additions of the soluble protein yield equal increments of stimulation. These results are consistent with the suggestion that the soluble protein may be a reactant in the oxidative process. Methyl sterol oxidase is inhitited in vitro by cholesterol, several oxygenated sterols, and cholesterol esters. The extent of inhibition is much greater when the soluble protein is present in the incubation. The inhibition is competitive with respect to methyl sterol substrate; cholesterol succinate, a water-soluble ester, is strongly inhibitory, K'i (ester)/K'm(substrate) approximately 0.2. Since end product inhibition of methyl sterol oxidase may be produced by accumulation of cholesterol or cholesterol metabolites, the soluble protein may participate in regulation of the activity of some microsomal enzymes of cholesterol biosynthesis.

Preparation and properties of a cell-free system from rat skin that catalyzes sterol biosynthesis.

Homogenates of epidermis from rat skin were centrifuged at 10,000 X g for 20 min. The supernatant fraction ("whole homogenate") catalyzed the demethylation of lanosterol (C(30)) to yield C(27)-sterols. The rate of reaction was measured by the rate of release of (14)CO(2) from the 4-methyl group of lanosterol. Conditions for maximal rates of demethylation were established. Addition of increasing amounts of washed microsomes to a constant amount of substrate resulted in additional release of (14)CO(2), but the release was not proportional to the amount of microsomes. Incubation with increasing amounts of microsomes treated with Triton WR-1339 yielded a proportional rate of release of (14)CO(2). The Triton-treated microsomes were frozen and stored without loss of activity. The rate of formation of (14)CO(2) was constant up to 1 hr of incubation with both Triton-treated microsomes and whole homogenate, for which the K(m) for lanosterol was 5.0 and 3.0 X 10(-5) M, respectively. Other 4-gem-dimethyl sterols were competitive inhibitors, K(i)', 2.0 and 5.5 X 10(-5) M. The enzyme system was inhibited by arsenite. 24,25-Dihydrolanosterol, 24,25-dihydrolanostenone, and squalene were demethylated by the homogenate. The whole homogenate catalyzed the incorporation of mevalonic acid, but not acetic acid, into squalene and sterols. The enzymatic properties of the sterol synthetic system from skin resemble those of similar preparations from rat liver.