Purification and characterization of a cytochrome P450 isozyme catalyzing lanosterol 14 alpha-demethylation (P45014DM) in hamster liver.
To characterize cholesterol synthesis in Syrian golden hamster, an isozyme of cytochrome P450 lanosterol 14 alpha-demethylase (P45014DM), which catalyzes the initial step in the biosynthesis of cholesterol from lanosterol, was purified and its mode of induction by microsomal enzyme inducers was characterized. P450450DM was purified from hamster livers by chromatography using aminooctyl-Sepharose CL-6B columns, to a specific content of 12.8 nmol/mg-protein. The purified protein displayed a single band on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 52,000. The absorption spectra of the oxidized form of the purified protein showed a Soret peak at 417 nm in a low-spin state and a Soret peak of reduced CO-binding complex at 448 nm. In a reconstituted system, the purified protein catalyzed 14 alpha-demethylation of 24,25-dihydrolanosterol (1.58 nmol/min/nmol-P450), although it did not show any activities toward testosterone and 7-ethoxyresorufin, marker substrates of other P450 families. Immunoblot analysis using an antibody against porcine P45014DM, which inhibited the activity of lanosterol 14-alpha-demethylation in the hamster liver microsomes, demonstrated that the level of this isozyme protein was markedly decreased in dexamethasone-treated hamster livers. This was accompanied by a decrease in the enzyme activity. In contrast, the levels and the activity in the phenobarbital- and 3-methylcholanthrene-treated hamsters were almost equal to that in the untreated animals.
Sekigawa
Y
Y
Department of Pharmaceutical Sciences, National Institute of Public Health, Tokyo, Japan.
Fukuhara
M
M
Sonoda
Y
Y
Sato
Y
Y
D000818:Animals; D002384:Catalysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D002784:Cholesterol / Q000096:biosynthesis; D002845:Chromatography; D006224:Cricetinae; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D015151:Immunoblotting; D007810:Lanosterol / Q000378:metabolism*; D008297:Male; D008647:Mesocricetus; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D010634:Phenobarbital / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D013053:Spectrophotometry; D058886:Sterol 14-Demethylase
D007810:Lanosterol; D008748:Methylcholanthrene; D003907:Dexamethasone; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D010088:Oxidoreductases; D058886:Sterol 14-Demethylase; D010634:Phenobarbital