Comparative study of cytochrome P-450 in liver microsomes: A form of monkey cytochrome P-450, P-450-MK1, immunochemically cross-reactive with antibodies to rat P-450-male

doi:10.1016/0006-2952(89)90050-6

Biochemical Pharmacology

Volume 38, Issue 2, 15 January 1989, Pages 361-365

https://doi.org/10.1016/0006-2952(89)90050-6 Get rights and content

Abstract

Cytochrome P-450, designated as P-450-MK1, which is cross-reactive with antibodies to rat P-450-male, was purified to an electrophoretical homogeneity from liver microsomes of the untreated male crab-eating monkey.

The molecular weight of P-450-MK1 was estimated to be 50,000 by sodium dodecyl sulfate-poly-acrylamide gel electrophoresis (SDS-PAGE). The oxidized form of P-450-MK1 showed a peak at 418 nm, indicating that this cytochrome is in a low spin state. The carbon monoxide-bound reduced form showed a peak at 451 nm. The first 22 amino acid residues of the NH₂-terminal sequence of P-450-MK1 was fairly homologous to those of P-450-male (75% identity, not including unidentified amino acid residues).

Unlike the P-450-male, P-450-MK1 did not exhibit catalytic activities for testosterone 2α- and 16α-hydroxylations and catalyzed testosterone 6β-hydroxylation. It is, therefore, suggested that although the spectral and immunochemical properties and the N-terminal amino acid sequence of P-450-MK1 were similar to those of P-450-male, the physiological functions of P-450-MK1 may be somewhat different from those of P-450-male.

Comparison of the physico-chemical properties of P-450-MK1 with those of P-450-D1 and P-450-HM2, which are cross-reactive with anti-P-450-male antibodies, purified from liver microsomes of dogs and humans, respectively, are also discussed.

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We isolated a new form of cytochrome P450 (P450) which was able to catalyze S-mephenytoin 4′-hydroxylation from hepatic microsomes of cynomolgus monkeys. The final preparation (referred to as P450 CMLd) was apparently homogenous judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the estimated minimum molecular weight of this protein was 53 kDa. The N-terminal amino acid sequence of P450 CMLd (identified 16 residues) was identical with that of protein encoded by P450 2C9 cDNA. P450 CMLd was cross-reactive with both antibodies raised against P450 2C11 and P450 2C9 which were purified from hepatic microsomes of male rats and humans, respectively. In hepatic microsomes of cynomolgus monkeys, both antibodies recognized two proteins showing different mobilities on SDS-PAGE (50 and 53 kDa). P450 CMLd was a good catalyst for S-mephenytoin 4′-hydroxylation in a reconstituted system. Anti-P450 2C9 antibody inhibited the activity of S-mephenytoin 4′-hydroxylase, but not the activities of R-mephenytoin 4′-hydroxylase and R- and S-mephenytoin N-demethylases in liver microsomes from cynomolgus monkeys. From these lines of evidence we conclude that P450 CMLd is classified into the P450 2C subfamily and acts as one of the S-mephenytoin 4′-hydroxylases in hepatic microsomes of cynomolgus monkeys.
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A panel of 30 monoclonal antibodies against rat hepatic microsomal cytochrome P450h (2C11) has been produced, purified, and characterized. A broad range of reactivities was observed when 13 purified rat cytochrome P450 isozymes were tested for epitope relatedness in a noncompetitive enzyme-linked immunosorbent assay or on immunoblots. Several antibodies were antigen-specific, others reacted with additional members of the 2C subfamily, and other monoclonal antibodies recognized cytochromes P450 from the 2E, 2B, 2A, and 1A subfamilies. Cytochromes P450p (3A1) and P4501 (3A2) did not react with any of the antibodies. A minimum of seven spatially distinct epitopes on cytochrome P450h were defined by the panel of antibodies. Immunoblot analysis of rat microsomes illustrated the male specificity of cytochrome P450h expression which extended to extrahepatic tissues including kidney and lung. A survey of various species by immunoblot analysis with several antibodies revealed little if any epitope relatedness among microsomal proteins from rats, mice, rabbits, hamsters, squirrel monkeys, guinea pigs, or humans. All of the antibodies were screened as potential inhibitors of cytochrome P450h-mediated testosterone hydroxylation in a reconstituted system. Although most of the antibodies were noninhibitory, greater than 70% inhibition of 2a-and 16a-hydroxylation of testosterone was observed with selected antibodies. These inhibitory antibodies gave similar results when benzphetamine-demethylation was evaluated in the reconstituted system. The inhibitory antibodies were then used to assess the role of cytochrome P450h in microsomal benzphetnmine N-demethylation, since this isozyme exhibits high catalytic activity for this substrate. Only 20-25% inhibition of benzphetamine metabolism was attained in microsomal preparations from adult male rats, and the antibodies did not influence the microsomal catalytic activity of immature males or females or adult females. Thus, despite the high level of expression of cytochrome P450h in microsomes from adult male rats and the high catalytic activity of the purified protein for benzphetamine, this isozyme contributes only a small portion of the metabolism of this substrate in microsomes.
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Two forms of cytochrome P450, referred to as P450 CMLb and P450 CMLc, were purified and characterized from hepatic microsomes of untreated cynomolgus monkeys (Macaca irus). The final preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The minimum molecular weights of P450 CMLb and P450 CMLc estimated from the mobilities on the gel were 48 and 50 kDa, respectively. The N-terminal amino acid sequences of P450 CMLb and P450 CMLc (first 13 residues) were identical, respectively, with those of P450 FI isolated from baboons and P450 MK-2, which has been characterized as a P450 3A enzyme in cynomolgus monkeys. P450 CMLb showed coumarin 7-hydroxylation and 7-ethoxycoumarin O-deethylation activities. This P450 enzyme reacted with anti-P450 2A6 antibody and acts as a coumarin 7-hydroxylase in hepatic microsomes. On the basis of these results P450 CMLb was classified into the 2A subfamily. P450 CMLb is expressed constitutively as one of the minor forms of P450 in liver microsomes of untreated cynomolgus monkeys and is inducible by phenobarbital. P450 CMLc reacted with anti-P450 3A4 antibody and hydroxylated testosterone at the 6β-position. The testosterone 6β-hydroxylation activities in hepatic microsomes of cynomolgus monkeys, common squirrel monkeys, and humans were strongly inhibited by the anti-P450 CMLc antibody. These results demonstrate that this P450 enzyme is in the P450 3A subfamily.
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Comparative study of cytochrome P-450 in liver microsomes: A form of monkey cytochrome P-450, P-450-MK1, immunochemically cross-reactive with antibodies to rat P-450-male

Abstract

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