Stoichiometry of 4-methyl sterol oxidase of rat liver microsomes.
The stoichiometry of 4-methyl sterol oxidase has been investigated by concurrent assays of rates of oxygen consumption, oxidation of reduced pyridine nucleotide, and formation of steroid 4alpha-oic acid, which is the oxidized product of attack of 4-methyl sterol precursors of cholesterol. The basal, steroid-independent rates of oxidation of alpha-NADH and alpha-NADH-dependent oxygen consumption by rat liver microsomes are about 10 to 15% of the rates observed with beta-NADH. Thus, alpha-NADH is substituted for beta-NADH; alpha-NADH oxidation is observed spectrophotometrically. The slow rate of oxygen consumption is measured accurately with a galvanic oxygen electrode that is attached to an offset amplifier. For maximal velocity, 4alpha-hydroxymethyl-5alpha-cholest-7-en-3beta-ol is the steroid substrate, and oxidase activity is induced 2-fold with a dietary bile acid sequestrant. Under these conditions, accurate measurements are obtained for substrate-dependent increments, which are equal to or greater than basal, substrate-independent rates. For each equivalent of hydroxymethyl group oxidized to carboxylic acid, 2 eq each of oxygen and alpha-NADH are consumed. Thus, the stoichiometry is consistent with that expected for two sequential attacks of the 4alpha-hydroxymethyl group by an external mixed function oxidase. In addition to establishing the stoichiometry of the 4-methyl sterol oxidase, the results further demonstrate that the steroidal 4alpha-carboxylic acid is formed from the hydroxymethyl intermediate by catalysis of a mixed function oxidase rather than dehydrogenases.
D000818:Animals; D002792:Cholestyramine Resin / Q000494:pharmacology; D003486:Cyanides / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D009243:NAD; D009249:NADP; D010101:Oxygen Consumption; D011092:Polyethylene Glycols / Q000494:pharmacology; D051381:Rats; D013261:Sterols; D013329:Structure-Activity Relationship
D003486:Cyanides; D013261:Sterols; D009243:NAD; D002792:Cholestyramine Resin; D011092:Polyethylene Glycols; D009249:NADP; D006899:Mixed Function Oxygenases