J. Biochem, 1988, Vol. 104, No. 3 481-487
© 1988 Japanese Biochemical Society
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Constitutive Testosterone 6ß-Hydroxylase in Rat Liver
*Laboratory of Chemistry, Osaka City University Medical School Abeno-ku, Osaka, Osaka 545
**Laboratory of Molecular Biology, Suntory Institute for Biomedical Research Shirnamoto-cho, Mishima-gun, Osaka 618
1To whom correspondence should be sent
The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119–132) had high testosterone 6ß-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1 : 1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidyl choline, P450 PB-1 had little 6ß-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4ą5.6% (meanąSD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r=0.925) between the P450 PB-1 level and testosterone 6ß-hydroxylase activity in rat liver microsomes. Furthermore, testosterone 6ß-hydroxylation activity of hepatic microsomes was completely inhibited by anti-P450 PB-1 antibody. The conclusion is that P450 PB-1 is the major or sole constitutive catalyst of testosterone 6ß-hydroxytase in rat liver.
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