title: Side chain hydroxylation of C27-steroids and vitamin D3 by a cytochrome P-450 enzyme system isolated from human liver mitochondria.
abstract: The present study was undertaken to obtain information on the involvement of cytochrome P-450 in the 26-hydroxylation on bile acid intermediates and in the 25-hydroxylation of vitamin D3 in human liver mitochondria. Cytochrome P-450 was solubilized from human liver mitochondria and purified two times to a specific content of 0.125 nmol per mg protein. Furthermore, a ferredoxin was isolated from the mitochondria and partly purified. This iron-sulfur protein had properties similar to bovine adrenal ferredoxin. A mitochondrial NADPH-ferredoxin reductase was also isolated and purified to homogeneity. This enzyme was a flavoprotein with properties very similar to the bovine adrenal NADPH-ferredoxin reductase. The cytochrome P-450 preparation catalyzed 26-hydroxylation of C27-steroids and 25-hydroxylation of vitamin D3 when reconstructed with NADPH, the ferredoxin and the ferredoxin reductase. With different substrates the following turnover numbers (nmol product X nmol P-450(-1) X min-1) were found: cholesterol, 8; 5-cholestene-3 beta, 7 alpha-diol, 10; 7 alpha-hydroxy-4-cholesten-3-one, 23; 7 alpha, 12 alpha-dihydroxy-4-cholesten-3-one, 27; 5 beta-cholestane-3 alpha, 7 alpha-diol, 28; 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol, 41; and vitamin D3, 0.16. The hydroxylation reactions were inhibited by CO and metyrapone. The human liver mitochondrial ferredoxin and ferredoxin reductase could be replaced by adrenal ferredoxin and adrenal ferredoxin reductase without reduction of activity, but they could not be replaced by microsomal NADPH-cytochrome P-450 reductase. It is concluded that human liver mitochondria contain cytochrome P-450 involved in the oxidation of the side chain of C27-steroids and vitamin D3.
mesh_terms: D000328:Adult; D000818:Animals; D002762:Cholecalciferol / Q000378:metabolism*; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification / Q000378:metabolism*; D005287:Ferredoxin-NADP Reductase / Q000378:metabolism; D006801:Humans; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology; D008930:Mitochondria, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism; D051381:Rats; D012995:Solubility; D013250:Steroid Hydroxylases / Q000378:metabolism*; D013256:Steroids / Q000378:metabolism; D014316:Tritium
chemical_list: D013256:Steroids; D014316:Tritium; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D005287:Ferredoxin-NADP Reductase; D009251:NADPH-Ferrihemoprotein Reductase