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.
D000818:Animals; D003600:Cytosol / Q000502:physiology; D004789:Enzyme Activation; D007700:Kinetics; D008099:Liver / Q000502:physiology*; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013256:Steroids / Q000494:pharmacology; D013261:Sterols; D013329:Structure-Activity Relationship
D011506:Proteins; D013256:Steroids; D013261:Sterols; D013250:Steroid Hydroxylases