Cholesterol biosynthesis from lanosterol: development of a novel assay method and characterization of rat liver microsomal lanosterol delta 24-reductase.
The membrane-bound sterol delta 24-reductase (24-reductase) catalyses anaerobic reduction of the 24(25)-enes of lanosterol and other obligatory intermediates of cholesterol biosynthesis from lanosterol. A novel assay method and properties of the 24-reductase are described. More than a 120-fold induction of the 24-reductase activity was achieved by feeding rats a diet containing 5% cholestyramine plus 0.1% lovastatin in chow and by modulating diurnal variation. With this enzyme induction condition, lanosterol was converted efficiently into dihydrolanosterol in both intact hepatic microsomes and freshly isolated hepatocytes only when either miconazole or CO was added to inhibit 14 alpha-demethylation of lanosterol. AR45 cells, which are deficient in 14 alpha-methyl demethylase (14 alpha-DM), exhibit lanosterol 24-reductase activity without addition of either CO or miconazole. Conversely, inhibition of the 24-reductase was not required for the expression of 14 alpha-DM activity. Studies on the substrate specificities for the 24-reductase using different 24(25)-enes showed that the most reactive substrate was 5 alpha-cholesta-7,24-dien-3 beta-ol, which exhibited a maximal 18-fold higher kcat than that of lanosterol without the aid of the 14 alpha-DM inhibitor. In addition, both the kinetic behaviour of lanosterol substrate in relation to the 24-reductase and a non-competitive inhibition mode of U18666A (Ki 0. 157 microM) as well as Triparanol (Ki 0.523 microM), two well-known 24-reductase inhibitors, were determined. On the basis of our new findings on the preferred substrate and on the negative effect of 14 alpha-DM on the 24-reductase, we suggest that C-24 reduction of sterols takes place straight after sterol delta 8-->7 isomerization of zymosterol, which occurs several steps after C-32 demethylation of lanosterol in the 19-step pathway of cholesterol biosynthesis from lanosterol.
Bae
S H
SH
Department of Biochemistry and Bioproducts Research Center, College of Science, Yonsei University, Seoul, Korea.
Paik
Y K
YK
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D002784:Cholesterol / Q000096:biosynthesis*; D002792:Cholestyramine Resin / Q000008:administration & dosage; D002940:Circadian Rhythm; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004032:Diet; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D007810:Lanosterol / Q000378:metabolism*; D008297:Male; D008780:Methyltransferases / Q000096:biosynthesis / Q000737:chemistry* / Q000187:drug effects; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D058886:Sterol 14-Demethylase; D013379:Substrate Specificity
C548417:Cyp51 protein, rat; D002792:Cholestyramine Resin; D007810:Lanosterol; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D008780:Methyltransferases; C022670:delta 24-sterol methyltransferase