Abstract
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.
Highlights
The slow rate of oxygen consumption is measured accurately with a galvanic oxygen electrode that is attached to an offset amplifier
In addition to the oxidative attack of the methyl group (Reaction A of Equation l), reduced pyridine nucleotide and oxygen are required for the further metabolism of the resulting 4cu-hydroxymethyl steroid to 4a-oic acid (Reactions B and C of Equation 1) (4)
Carboxylic acid is formed under aerobic conditions, and alterations in the rates of mixed function oxidative attack of methyl sterol substrates yield similar alterations in the rates of mixed function oxidative attack of hydroxymethyl sterol substrates, substantiation of this suggestion has been delayed because measurement of the stoichiometry of consumption of oxygen and reduced pyridine nucleotide by this relatively slow microsomal oxidase has been impossible until this time
Summary
Of 4-Methyl Sterol Oxidase of Rat Liver (Received for publication, December 5, 1974). In addition to the oxidative attack of the methyl group (Reaction A of Equation l), reduced pyridine nucleotide and oxygen are required for the further metabolism of the resulting 4cu-hydroxymethyl steroid to 4a-oic acid (Reactions B and C of Equation 1) (4). Carboxylic acid is formed under aerobic conditions, and alterations in the rates of mixed function oxidative attack of methyl sterol substrates yield similar alterations in the rates of mixed function oxidative attack of hydroxymethyl sterol substrates, substantiation of this suggestion has been delayed because measurement of the stoichiometry of consumption of oxygen and reduced pyridine nucleotide by this relatively slow microsomal oxidase has been impossible until this time. Increments of steroid-dependent changes in concentrations of oxygen and reduced pyridine nucleotide could not be observed accurately with the use of P-NADH as the source of reducing equivalents for the external mixed function oxidase These results not onlv describe methods suitable for the measurement of very slow microsomal mixed function oxidases and confirm that methyl sterol oxidase is an external mixed function oxidase, but this evidence demonstrates that metabolism of the 4cr-hydroxymethyl sterol intermediate of cholesterol biosynthesis is, catalyzed by a mixed function oxidase and not by microsomal dehydrogenases
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