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.
D000818:Animals; D002784:Cholesterol / Q000096:biosynthesis*; D002852:Chromatography, Ion Exchange; D003573:Cytochrome b Group / Q000378:metabolism; D015786:Cytochromes b5; D007810:Lanosterol / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D051381:Rats; D011919:Rats, Inbred Strains; D013997:Time Factors
D003573:Cytochrome b Group; C033144:lathosterol delta-5-dehydrogenase; D009243:NAD; D007810:Lanosterol; D015786:Cytochromes b5; D002784:Cholesterol; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors