Microsomal enzymes of cholesterol biosynthesis from lanosterol. Purification and characterization of delta 7-sterol 5-desaturase of rat liver microsomes.

Microsomal delta 7-sterol 5-desaturase of cholesterol biosynthesis is a multienzyme system which catalyzes the introduction of the delta 5-bond into delta 7-cholestenol to form 7-dehydrocholesterol. The detergent-solubilized 5-desaturase has been purified more than 70-fold and resolved from electron carriers and other rat liver microsomal enzymes of sterol biosynthesis by chromatography on DEAE-Sephacel, CM-Sepharose, and immobilized cytochrome b5; the 5-desaturase had not been fully resolved from cytochrom b5 reductase in earlier work. A functional electron transport system for the 5-desaturase has been reconstituted by combining the purified 5-desaturase and electron carriers with egg phosphatidylcholine liposomes. Optimizations of conditions for reconstitution have been obtained; both cytochrome b5 and NADH-cytochrome b5 reductase serve as electron carriers. A pyridine nucleotide-dependent flavoprotein is required and the requirement can be satisfied with either purified cytochrome b5 reductase or cytochrome P-450 reductase. Cyanide and iron-chelators strikingly inhibit the 5-desaturase activity, thus suggesting that 5-desaturase is a metalloenzyme as are other well-characterized cytochrome b5-dependent oxidases. 5-Desaturase is resolved from 4-methyl sterol oxidase activity of cholesterol biosynthesis by chromatography on the immobilized cytochrome b5. This resolution of the two oxidases not only indicates that introduction of the delta 5-bond and oxidation of 4 alpha-methyl groups are catalyzed by different terminal oxidases, but resolution affords enzymes of sufficient purity to carry out reconstitution experiments. A novel assay based on substrate-dependent increments of oxidation of alpha-NADH has been developed for measurement of 5-desaturase activity. Measurement of stoichiometry of 5-desaturase demonstrates that for each equivalent of cis-desaturation of delta 7-cholestenol, 1 eq of NADH is consumed. Along with strict dependence upon oxygen, this observation confirms, as suggested by previous workers, that the 5-desaturation is catalyzed by a mixed function oxidase rather than a dehydrogenase.