Total enzymic synthesis of cholesterol from lanosterol. Cytochrome b5-dependence of 4-methyl sterol oxidase.
Methyl sterol oxidase of microsomal synthesis of cholesterol from lanosterol is a mixed-function oxidase that is dependent upon reduced pyridine nucleotide. The methyl sterol oxidase, as well as NADH-cytochrome c reductase, in intact rat liver microsomes are inhibited by anti-cytochrome b5 immunoglobulin, but NADPH-cytochrome c reductase is not affected. There is a decreased time lag prior to onset of reoxidation of steady state levels of reduced cytochrome b5 when 4-methyl sterol oxidase substrates are present. Trypsin treatment of microsomes destroys cytochrome b5 with loss of methyl sterol oxidase activity. Activity is restored by addition of purified cytochrome b5 to trypsin-treated microsomes. Initial attempts to solubilize and purify 4-methyl sterol oxidase have been only partially successful due to the extreme lability of the oxidase. However, DEAE-cellulose column chromatography of a detergent extract of microsomes yields a fraction that contains the oxidase, lipids, and NADH-cytochrome b5 reductase but is free of cytochrome b5. Oxidation of 4 alpha [30-3H] methyl-5 alpha-cholest-7-en-3 beta-ol by methyl sterol oxidase in this isolated fraction can be fully restored by the addition of purified liver microsomal cytochrome b5. These results strongly support the suggestion that membrane-bound cytochrome b5 of rat liver microsomes is an obligatory electron carrier from NADH to 4-methyl sterol oxidase.
D000818:Animals; D002250:Carbon Radioisotopes; D002784:Cholesterol / Q000096:biosynthesis*; D003580:Cytochromes / Q000302:isolation & purification / Q000378:metabolism*; D015786:Cytochromes b5; D007700:Kinetics; D007810:Lanosterol / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D010088:Oxidoreductases / Q000378:metabolism*; D051381:Rats; D014316:Tritium
D002250:Carbon Radioisotopes; D003580:Cytochromes; D014316:Tritium; D007810:Lanosterol; D015786:Cytochromes b5; D002784:Cholesterol; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; C027532:methylsterol monooxygenase