Abstract
Rat hepatic cytochrome P-450 isoenzyme 2c, purified to homogeneity from uninduced, adult rat liver (Waxman, D.J., Ko, A., and Walsh, C. (1983) J. Biol. Chem. 258, 11937-11947), was shown to exhibit a unique NH2-terminal amino acid sequence as well as distinctive peptide maps and immunochemical properties when compared to seven other purified rat liver P-450 isoenzymes. P-450 2c was an efficient monooxygenase catalyst with several xenobiotic substrates; P-450 2c also catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone, androst-4-ene-3,17-dione and progesterone (total turnover = 7-9 min-1 P-450(-1) at 25 microM steroid substrate) with the ratio of 2 alpha to 16 alpha hydroxylation varying from less than or equal to 0.02 to 1.6 depending on the steroid's C-17 substituent. Six different microsomal steroid hydroxylase activities characteristic of purified P-450 2c and sensitive to specific inhibition by anti-P-450 2c antibody were induced at puberty in male but not female rat liver. Microsomal steroid hydroxylations catalyzed by other P-450 isoenzymes exhibited age and sex dependencies distinct from those of the P-450 2c-mediated activities. Immunochemical analyses confirmed that this sex dependence and developmental induction reflected alterations in P-450 2c polypeptide levels. Attempts to chromatographically detect P-450 2c in either immature male or adult female microsomes were unsuccessful and led to purification of P-450 2d (female), a catalytically distinct and female-specific form. Peptide mapping and immunochemical analyses suggested significant structural homologies between the two sex-specific isoenzymes, P-450 2c and P-450 2d (female). A significant suppression of P-450 2c levels (up to 70-80%) was observed upon administration of several classical P-450 inducers. These studies establish that P-450 2c corresponds to the male-specific and developmentally-induced steroid 16 alpha-hydroxylase of rat liver and suggest that the expression of P-450 2c versus P-450 2d (female) may provide a biochemical basis for the sex differences characteristic of rat liver xenobiotic metabolism.
Highlights
Chem. 258, 11937-11947), was shown toexhibit a alyzes the oxidative metabolism of structurally diverse carcinunique NHz-terminal amino acid sequence as well as ogens, pollutants, and drugs as well as endogenous steroids, distinctive peptide maps andimmunochemical properties when compared to seven other purified rat liver P-450 isoenzymes
Steroidhormones havebeenproposed to serve as physiological substrates for liver microsomal P-450, in part based on th1e0-100-fold higher affinities of P-450 for gonadal steroids as compared tloipophilic foreign compounds (Kuntzman et al, 1965; Orrenius and Lisboa, 1969)
The capacity of mammalian liver for steroid hormone hydroxylation is often dependent on sex suggesting that at least some of the liver microsomal P-450 isoenzymes active in steroid hydroxylation might be expressed in a sex-dependent fashion
Summary
450; 1‘”steroid 5n-reductase,NADPH; ~4-3-oxosteroid-5a-oxidoreductase; PBS, 10 mM KP, (pH 7.4), 0.9% NaC1; PB, phenobarbital; SDS gels, sodium dodecyl sulfate polyacrylamide gels; TLC, thinlayer chromatography; HPLC, high-performance liquid chromatography; T or testosterone, 17~-hydroxyandrost-4-ene-3-onAe;or androstenedione, androst-4-ene-3,17-dioneP; or progesterone, pregn-4-. P-4,502c Purification-P-450 2c was purified from uninduced, 1014-week-old (dependingonthepreparation) male rat liver microsomes by cholate-solubilization,polyethylene glycol 6000 (Baker) precipitation,andWhatmanDE52ion-exchangechromatography using the general methods described previously for purification of P450s PB-4 and PB-5 (Waxman and Walsh, 1982). Fractions judged 295% pure on SDS gels (specific content = 12-14 nmol of P-450/mg of protein) were pooled, dialyzed against 10 mM KPi (pH 7.4), 20% glycerol (v/ v), 0.1 mM EDTA, and rebound to hydroxylapatite for cholate exchange followed by dialysis as described for P-450 PB-1 (Waxman and Walsh, 1983). For affinity purification of antibodies, liver microsomes (400 mg; isolated from either mature female, mature male or phenobarbitalinduced immature male rats, as appropriate) were solubilized with 3.3% Emulgen 911, fractionated withpolyethylene glycol 6000 (515%) covalently immobilized onto a carboxyl-terminal agarose (12 ml settled Affi-Gel 202 resin; Bio-Rad) by incubationwith 1-. Other Methods-SDS-gel electrophoresis, NHz-terminal sequence analysis, partial proteolysis peptide mapping, Lowry protein determinations, and other analytipcraolcedures were as detailed previously (Waxman and Walsh, 1982,1983)
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