Abstract
To characterize cholesterol synthesis in Syrian golden hamster, an isozyme of cytochrome P450, lanosterol 14α-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-4B, hydroxylapatite, DEAE-5PW, and CM-Sephrose 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 howed 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 14α-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 14α-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.
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Abbreviations
- anti-P45014DM :
-
anti-porcine P45014DM antibody
- DHL:
-
24,25 dihydrolanosterol (lanost-8-3n-3β-ol)
- P45014DM :
-
P450 isozyme cat alyzing lanosterol 14α-demethylation
- PAGE:
-
polyacrylamide gel electrophoresis
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Sekigawa, Y., Fukuhara, M., Sonoda, Y. et al. Purification and characterization of a cytochrome P450 isozyme catalyzing lanosterol 14α-demethylation (P45014DM) in hamster liver. Lipids 30, 1067–1073 (1995). https://doi.org/10.1007/BF02536606
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DOI: https://doi.org/10.1007/BF02536606