Properties will be studied of a homogeneous liver microsomal cytochrome P-450 which oxygenates and removes C-32 of lanosterol as formic acid. The mechanism of the reaction will be investigated with [32-14C], [32-3H], and [15Alpha-3H]lanosterol all of which are probes for enzyme activity. 15Alpha and 15Beta-Hydroxy-14-methylsterols will be investigated as possible intermediates. Formation of formate from lanosterol and its 14-hydroxymethyl and 14-formyl analogs will be studied in an atmosphere of 18O2 to determine the fate of oxygen in each step of formate removal. The results of these investigations will clarify the mechanism of a similar methyl group removal in the aromatase reaction and in other P-450 catalyzed C-C cleavage reactions, i.e., cholesterol side-chain cleavage to form pregnenolone, and C21-steroid lyase to form testosterone. An intimate knowledge of 14Alpha-demethylase may help to design new antifungal agents, since many compounds that are potent inhibitors of 14Alpha-demethylase are effective antigungal agents in medicine and agriculture. The properties and mechanism will be studied of a homogeneous non-heme iron oxygenase which converts the Alpha-methyl group at C-4 of lanosterol to a carboxylic acid. An oxygen-dependent cyanide-sensitive enzyme will be purified which catalyzes formation of the 5,6-double bond of cholesterol. With antibodies prepared against the above three purified enzymes, we shall clone their genes by available recombinant DNA techniques. The sequence of the DNAs will be used to determine the amino acid sequence of the enzymes for a comparison with that of stearoyl CoA desaturase and of other microsomal oxygen activating enzymes sequenced elsewhere. This information should yield valuable genetic and evolutionary relationships.
