Constitutive Forms Of Cytochrome P-450 Research Articles

Expression of CYP2M1, CYP2K1, and CYP3A27 in brain, blood, small intestine, and other tissues of rainbow trout.

Expression of five constitutive forms of cytochrome P450 [(LMC1 (CYP2M1), LMC2 (CYP2K1), LMC3, LMC4, and LMC5 (CYP3A27)] in selected tissues from sexually immature 2-year old female and male rainbow trout (Oncorhynchus mykiss) were examined at the translational level by Western blot using polyclonal antibodies raised in rabbits against those purified trout hepatic P450s. Tissues examined were from brain, liver, muscle, blood, head kidney, trunk kidney, upper intestine, stomach, heart, and gonad (ovary or testis). The results showed that the liver was the major organ for expression of all the trout P450s studied. Trunk kidney was the secondary expression site except for LMC5. Selective translational expression of these P450 isoforms or similar proteins was observed for LCM1 and LMC5 in brain; for LMC2 and LMC5 in female upper intestine; and for LMC2 in blood plasma of the fish studied under the experimental and sampling conditions.

Purification and characterization of three constitutive cytochrome P-450 isoforms from bovine olfactory epithelium.

Three constitutive forms of cytochrome P-450 (P-450s) were isolated from olfactory microsomes of cattle. The purified P-450s, designated P-450bov1, P-450bov2 and P-450bov3, were electrophoretically nearly homogeneous by SDS/PAGE and their apparent relative molecular masses were estimated to be 50000, 53000 and 51000 respectively. As indicated by several criteria including the N-terminal sequence and absorption spectra, the three olfactory forms of P-450 were distinct from each other and from all the other P-450s currently known in cattle. P-450bov1 and P-450bov2 were purified in the low-spin state, whereas P-450bov3 was in the high-spin state. Studies to evaluate, by Western blot analysis, the reactivity of these purified P-450s with antibodies raised against rat hepatic P-450 2E1, 2B, 1A and 3A and rabbit olfactory P-450NMa and P-450NMb showed that P-450bov3 strongly cross-reacted with anti-P-450NMb IgG, and P-450bov1 moderately with anti-P-450NMa IgG. As determined by immunoblots, P-450bov1 and P-450bov3 represented a great portion of the total olfactory P-450. In a reconstituted system with NADPH:cytochrome P-450 reductase and phospholipids, P-450bov1 was more active in the metabolism of xenobiotic compounds (i.e. O-de-ethylation of ethoxycoumarin and N-demethylation of hexamethylphosphoramide) than towards endogenous substrates (testosterone and progesterone). Conversely, P-450bov3 metabolized the xenobiotics at lower rates but exhibited total oxidation rates of the above sex hormones higher than those of P-450bov1. From the comparison of the catalytic, immunochemical and structural properties, it was inferred that P-450bov1 and P-450bov3 are the bovine orthologues of P-450NMa (2A) and P-450NMb (2G1) respectively, the only two olfactory P-450s previously purified from rabbit. P-450bov2, which showed low activity toward some exogenous and endogenous compounds, represents a novel purified olfactory hemoprotein possibly belonging to the 3A subfamily. These results are consistent with a specific presence of catalytically and structurally similar P-450s, at least for the major ones, in the olfactory mucosa of mammals.

Regulation of Cytochrome P450 2C11 (CYP2C11) Gene Expression by Interleukin-1, Sphingomyelin Hydrolysis, and Ceramides in Rat Hepatocytes

Interleukin-1 triggers the down-regulation of several hepatic cytochrome P450 gene products, but the cellular signaling pathways involved are not known. We have examined the role of sphingomyelin hydrolysis to ceramide in the suppression of CYP2C11, a major constitutive form of cytochrome P450, by interleukin-1. Treatment of rat hepatocytes cultured on matrigel with interleukin-1 beta caused a rapid turnover of sphingomyelin and an increase in cellular ceramide, with no change in cellular phosphatidylcholine. The ceramide was composed mainly of a D-erythro-sphingosine backbone, suggesting that it was derived from sphingolipid hydrolysis rather than from increased de novo synthesis. Treatment of the cells with either N-acetyl-D-erythro-sphingosine (C2-ceramide) or bacterial sphingomyelinase suppressed the expression of CYP2C11 and induced the expression of the interleukin-1-responsive alpha 1-acid glycoprotein mRNA. In contrast, the acute-phase gene beta-fibrinogen, which is induced by interleukin-6 but not by interleukin-1, did not respond to C2-ceramide. N-Acetyl-D-erythro-sphinganine mimicked the effect of C2-ceramide on CYP2C11, but not on alpha 1-acid glycoprotein expression. These results are consistent with a role for ceramide or a related sphingolipid in mediating the down-regulation of CYP2C11, the induction of alpha 1-acid glycoprotein, and perhaps other cellular effects of interleukin-1 in hepatocytes.

Channel catfish liver monooxygenases: Immunological characterization of constitutive cytochromes P450 and the absence of active flavin-containing monooxygenases

Multiple drugs and pesticides are used in the aquaculture of channel catfish in the Southeastern United States. However, little is known regarding the enzymatic metabolism of these chemicals in the fish. Western blots, utilizing polyclonal antibodies raised against five purified rainbow trout liver cytochrome P450 enzymes, revealed at least two protein bands that were approximately 50 kDa (CATL-1) and 53 kDa (CATL-2). Anti-trout LMC3 and LMC4 only hybridized with the 53 kDa protein, whereas anti-trout LMC1, LMC2, and LMC5 recognized both proteins. Cytochrome P450-catalyzed activities (testosterone and progesterone hydroxylases) associated with LMC1 and LMC5 were also found in catfish liver microsomes. These data suggest that at least two constitutive forms of cytochrome P450 are present in the liver of juvenile channel catfish. Western blots utilizing antibodies raised against rabbit-lung flavin-containing monooxygenases (FMO) showed hybridization with two proteins from rainbow trout liver microsomes, but no cross-reaction with nucrosomes from catfish liver. N, N,-Dimethylaniline N-oxidase and methimazole oxidase were observed in microsomes from trout, but were absent in catfish liver microsomes prepared in three different laboratories. Consequently, FMO do not appear to be present in liver microsomes from channel catfish or they are rapidly degraded during tissue homogenization.

Tissue-specific expression, induction, and inhibition through metabolic intermediate-complex formation of guinea pig cytochrome P450 belonging to the CYP2B subfamily

The tissue-specific expression and induction of P450 GP-1, a constitutive form of cytochrome P450 of the guinea pig classified into the CYP2B subfamily, were studied. Prior to these studies, a P450 form (P450 GP-1 PB) was purified from phenobarbital-treated guinea pigs and the properties were compared with those of the P450 GP-1. This form was judged to be the same as P450 GP-1 existing in untreated animals by comparisons of their N-terminal amino acid sequences, peptide maps, and affinities toward anti-P450 GP-1 antibody. Immunostaining of P450 GP-1 revealed that the lung and small intestine as well as the liver of untreated guinea pigs contain P450 GP-1, while none or only small amounts of this P450 form were observed in the kidney, heart, spleen, urinary bladder, and testis. The amount of liver P450 GP-1 protein expressed in untreated guinea pigs was estimated to be 19.4% of the total cytochrome P450 and this form was increased 1.7-fold by phenobarbital treatment. Similarly, intestinal P450 GP-1 was increased by phenobarbital treatment. However, lung P450 GP-1 was not increased by the treatment. It was also observed that the liver P450 GP-1 is induced with SKF-525A to the same extent as with phenobarbital. On the other hand, dexamethasone, p,p′-dichlorodiphenyltrichloroethane, and isosafrole showed no or only a weak ability to increase the liver P450 GP-1 content. The drug-metabolizing activities in the liver microsomes of SKF-525A-pretreated guinea pigs were lower than those in phenobarbital-treated animals, although the P450 GP-1 protein was induced equally by these treatments. The low activities of SKF-525A-treated animals in the drug metabolisms were attributed to the formation of the metabolic-intermediate complex between P450 GP-1 and SKF-525A metabolite. These results permitted us to conclude that the tissue specificity in the expression of guinea pig P450 belonging to the CYP2B subfamily and the inducibility with chemicals are similar to those of rat CYP2B1, although the constitutive expression of guinea pig liver P450 GP-1 is much higher than that of CYP2B1.

The effect of age on inducibility of various types of rat liver cytochrome P-450.

1. The content and specific activities of inducible cytochrome P-450 enzymes were determined in liver microsomes of rats of various ages after maximal induction with phenobarbital, isosafrole of 3-methylcholanthrene, and in untreated animals. 2. With age an increase in liver weight was observed both in untreated rats and in maximally induced ones; the microsomal protein content/g of liver decreased with age in untreated animals but not in induced ones. Total cytochrome P-450 content/mg microsomal protein remained unchanged with age in all experimental groups. 3. Immunologically detectable levels of cytochrome P4501A1/1A2 and 2B1/2B2 remain unchanged with age both in untreated animals and in maximally induced ones. 4. Several cytochrome P-450 activities showed an age-related decrease in untreated animals, but no change with age was observed in the activities of cytochrome P4501A1, 2A2 and 2B1/2B2 in rat liver microsomes. This indicates that ageing affects only the activity of some constitutive forms of cytochrome P-450 in male rats, but not the activity of inducible types of P-450. 5. Although previous results indicated decreased inducibility of the cytochrome P-450 mRNA levels with age, the present study clearly demonstrates that this is not reflected in decreased enzyme levels or activities after maximal induction. From this it is concluded that the decreased mRNA levels might rather be reflected in a decreased rate at which maximal induction can be achieved.

Decrease in a constitutive form of cytochrome P-450 by macrolide antibiotics

The effects of administration of macrolide antibiotics on cytochrome P-450 in liver microsomes of male rats were investigated. The macrolides tested were those with a 14-membered ring such as oleandomycin, troleandomycin, erythromycin and erythromycin estolate, and those with a 16-membered ring such as rokitamycin, leucomycin and josamycin. Cytochrome P-450-metabolite complex was detected with oleandomycin, troleandomycin, erythromycin and erythromycin estolate, whereas no such effect was observed with rokitamycin, leucomycin and josamycin. The content of uncomplexed cytochrome P-450 in liver microsomes remained unchanged with rokitamycin, leucomycin and josamycin, decreased with troleandomycin and oleandomycin, and increased with erythromycin and erythromycin estolate, indicating that oleandomycin, troleandomycin, erythromycin and erythromycin estolate also affect the amounts of other forms of cytochrome P-450. The administration of oleandomycin, troleandomycin, erythromycin and erythromycin estolate resulted in a dramatic decrease in the activities of testosterone 2 alpha- and 16 alpha-hydroxylases in liver microsomes. Supporting these results, a marked decrease (more than 75%) in the content of P-450-male, a major constitutive form of cytochrome P-450 in male rats, was noted with oleandomycin, troleandomycin, erythromycin and erythromycin estolate, while the decrease was rather small with rokitamycin and leucomycin. We conclude that the administration of the 14-membered ring macrolides may affect drug and steroid metabolism not only by formation of P-450-metabolite complex but also by decrease in the content of P-450-male.

Expression of a constitutive form of cytochrome P450 during rat-liver development and sexual maturation.

Cytochrome P450 cDNA clone designated pP450 IGC 1 has been previously isolated from a phenobarbital-induced rat liver cDNA library, characterized and proven to correspond to a constitutive cytochrome P450 sensitive to phenobarbital stimulation. IGC 1 pDNA, as well as a unique synthetic oligonucleotide probe, were used to investigate the mRNA levels at different developmental stages (fetal, male and female rats of various ages) in parallel with the study of the expression of the liver development and male phenotype markers: albumin, alpha-fetoprotein and alpha 2u globulin mRNAs. Cytochrome P450 IGC 1 mRNA responsiveness to phenobarbital administration was studied in animals of both sexes at different developmental stages. P450 IGC 1 mRNA was not detectable in fetuses nor in male or female rat liver before sexual maturation, becoming elevated at 45 days age, increasing up to 3 months and reaching relatively higher levels in the female liver than in the male. The concentrations of P450 IGC 1 mRNA closely paralleled the increases in alpha 2u globulin mRNA in male liver, as analyzed by dot and Northern blot hybridization. However, P450 IGC 1 was markedly induced by phenobarbital already at 20 days age both in males and females while alpha 2u globulin mRNA was not inducible before sexual maturation. It is concluded that the expression of cytochrome P450 IGC 1 mRNA is associated with the sexual maturation being differently modulated in the male and in the female rat liver. This cytochrome P450 isoenzyme may play an important physiological role in sex differential steroid metabolism and susceptibility to cytotoxic and genotoxic agents.

Regioselective hydroxylation of prostaglandins by constitutive forms of cytochrome P-450 from rat liver: Formation of a novel metabolite by a female-specific P-450

Previous studies demonstrated that liver microsomes from untreated rats catalyze the ω, ω-1, and ω-2 hydroxylation of prostaglandins [ K. A. Holm, R. J. Engell, and D. Kupfer (1985) Arch. Biochem. Biophys. 237, 477–489 ]. The current study examined the regioselectivity of hydroxylation of PGE 1 and PGE 2 by purified forms of P-450 from untreated male and female rat liver microsomes. PGE 1 was incubated with a reconstituted system containing cytochrome P-450 RLM 2, 3, 5, 5a, 5b, 6, or f4, NADPH- P-450 reductase, and dilauroylphosphatidylcholine in the presence or absence of cytochrome b 5. Among the P-450 forms examined, only RLM 5 (male specific), 5a (present in both sexes), and f4 (female specific) yielded high levels of PGE hydroxylation. With PGE 1, RLM 5 catalyzed solely the ω-1 hydroxylation and 5a catalyzed primarily the ω-1 and little ω and ω-2 hydroxylation. By contrast, f4 effectively hydroxylated PGE 1 and PGE 2 at the ω-1 and at a novel site. Based on retention on HPLC and on limited mass fragmentation, we speculate that this site is ω-3 (i.e., 17-hydroxylation). Kinetic analysis of PGE 1 hydroxylation demonstrated that the affinity of f4 for PGE 1 is approximately 100-fold higher than that of RLM 5; the K m values for f4, monitoring 19- and 17-hydroxylation of PGE 1, were about 10 μ m. Surprisingly, cytochrome b 5 stimulated the activity of RLM 5a and f4, but not that of RLM 5. Hydroxylation of PGE 2 by RLM 5 was at the ω, ω-1, and ω-2 sites, demonstrating a lesser regioselectivity than with PGE 1. These findings show that the constitutive P-450s differ dramatically in their ability to hydroxylate PGs, in their regioselectivity of hydroxylation, and in their cytochrome b 5 requirement.

Metabolic activation of a protein pyrolysate promutagen 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline by rat liver microsomes and purified cytochrome P-450.

The enzymatic activation of a promutagenic pyrolysate, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), was studied using the Ames mutagenesis test system. The enzyme catalyzing the mutagenic activation of MeIQx is mainly localized in the microsomal fraction. A large number of revertants was observed in the presence of hepatic microsomes obtained from 3-methylcholanthrene (3-MC)- or polychlorinated biphenyl (PCB)-treated rats but only a minimal number with the hepatic microsomes from untreated or phenobarbital (PB)-treated rats. In addition, the microsomal activation was reduced efficiently by known inhibitors of cytochrome P-450-mediated reactions such as 7,8-benzoflavone, ellipticine and flavone. Among five forms of purified rat cytochrome P-450, the highest sp. act. (no. of revertants induced/nmol cytochrome P-450) for the activation of MeIQx was observed with a high-spin form of cytochrome P-450, P-448-H, followed by the low-spin form, P-448-L, and to a lesser extent by PB-inducible forms, P-450b and P-450e. P-450-male, which is a main constitutive form of cytochrome P-450 in male rat livers, showed considerable catalysis for the mutagenic activation of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) and MeIQx. These results indicate that the metabolic activation of MeIQx is catalyzed mainly by two forms of cytochrome P-450, P-448-H and P-488-L, in the livers of PCB- or 3-MC-treated rats, but also that P-450-male may play an important role in the activation in livers of intact male rats.

Genotoxic and mutagenic activation of aflatoxin B 1 by constitutive forms of cytochrome P-450 in rat liver microsomes

Cytochrome P-450-mediated activation of aflatoxin B 1 (AFB 1) to genotoxic and mutagenic products which subsequently cause induction of an umu gene expression in Salmonella typhimurium TA1535/pSK1002 has been studied in a rat liver microsomal or reconstituted monooxygenase system. Liver microsomes from male Sprague-Dawley rats had a 1.5-fold higher activity to catalyze AFB 1 than did those from female rats. In addition, the activation was not increased in liver microsomes from rats pretreated with phenobarbital, 3-methylcholanthrene, a polychlorinated biphenyl mixture, or dexamethasone, suggesting that the constitutive forms of cytochrome P-450 have important roles for the activation of AFB 1 in rat liver microsomes. Using 15 forms of cytochrome P-450 purified from liver microsomes of untreated and phenobarbital- and 3-methylcholanthrene-treated rats, three isozymes from untreated male rats and one isozyme from untreated female rats were found to have high reactivities in metabolizing AFB 1 to genotoxic products. Cytochrome P-450 forms isolated from inducer-treated rats were relatively less active. The close correlation between induction of umu gene expression and mutagenicity with Ames/ S. typhimurium TA98 system by activated metabolites of AFB 1 in the reconstituted monooxygenase system suggested that the constitutive forms of cytochrome P-450 had major roles for genotoxic and mutagenic activation of AFB 1 in rat liver microsomes.

Hepatic metabolism of cyclodiene insecticides by constitutive forms of cytochrome P-450 from lower vertebrates

1. Multiple forms of cytochrome P-450 were separated from the hepatic microsomes of untreated male rats, pigeons ( Columbia livia), razorbills ( Alca torda), puffins ( Fratercula arctica), and rainbow trout ( Salmo gairdnerii), using anion exchange chromatography and DEAE-cellulose. 2. In some cases cytochrome P-450 forms were further purified on hydroxylapatite and carboxymethylsephadex columns. 3. Considerable differences in the distribution of forms between these five species were evident from elution profiles on DEAE cellulose, and on analysis of the cytochrome P-450 containing pools by SDS-PAGE. 4. The metabolism of two organochlorine compounds, aldrin § § Aldrin (HHDN) = 1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8 a-hexahydro-exo-1,4,-endo-5,8,-dimethanonaphthalene. and the dieldrin ∥ ∥ Dieldrin (HEOD) = 1,2,3,4,10,10-hexachloro-1,4,4 a,5,6,7, 8,8a-octahydro-6,7-epoxy-exo-1,4-endo-5,8-dimethanonaphthalene. analogue HCE, ¶ ¶ HCE = 1,2,3,4,9,9-hexachloro-1,4,4a,5,6,7,8,8a-octahydro-exo-7,8-epoxy-1,4-methanonaphthalene. were studied in (a) intact microsomes and (b) reconstituted systems containing cytochrome P-450, from each of the five species. 5. In spite of their close structural similarity, significant differences were found between the two substrates in the distribution of catalytic activity between the cytochrome P-450 isozymes of each species.

Separation of two constitutive forms of cytochrome P-450 active in aminopyrine N-demethylation from rabbit intestinal mucosa microsomes

Two constitutive forms of cytochrome P-450, designated P-450ib and P-450ic, were purified from intestinal mucosa microsomes of untreated rabbits. P-450ib and P-450ic have minimal molecular weights of 56 000 and 49 000, respectively, as determined by calibrated sodium dodecyl sulphate polyacylamide gel electrophoresis. The CO-reduced difference spectral maximum of cytochrome P-450ib is at 450 nm and P-450ic is at 451 nm. Both the cytochromes preferentially demethylate aminopyrine, benzphetamine and N, N-dimethylaniline in the presence of NADPH-cytochrome P-450 reductase. Cytochrome P-450ib has absorption maxima at 417, 535 and 573 nm in the oxidized form, indicating that this cytochrome is in a low-spin state. Ouchterlony double-diffusion studies show that cytochrome P-450ib does not cross-react with antisera against liver cytochrome P-450 LM2 purified from phenobarbital-treated rabbits, but P-450ic cross-reacts with spur formation. Unlike cytochrome P-450ib, P-450ic is very similar, if not identical, to liver cytochrome P-450 LM2 on the basis of its molecular weight, spectral properties, catalytic activities and immunochemical properties.

Isolation and characterization of two constitutive forms of microsomal cytochrome P-450 from a single bovine liver

Two constitutive forms of cytochrome P-450 isozyme were isolated from microsomes prepared from a single bovine liver. The two highly purified isozymes were electrophoretically homogeneous on SDS-polyacrylamide gel and their apparent minimum molecular weights were estimated to be 50 000 and 55 000. The isozyme of smaller molecular weight, designated cytochrome P-450A, and the one of larger molecular weight, designated cytochrome P-450B, were distinct proteins by the criteria, SDS-polyacrylamide gel electrophoresis, peptide maps, amino acid contents. To reveal the immunochemical relation between these two isozymes, antibodies to each isozyme was raised in rabbit. Antibodies to cytochrome P-450A gave a single precipitin line against its antigen in Ouchterlony double-diffusion plates, but did not cross-react against cytochrome P-450B. On the other hand, antibodies to cytochrome P-450B formed a single precipitin line with its antigen and did not show any cross-reactivity against cytochrome P-450B. These results indicate that two isozymes are immunochemically distinct. This conclusion was supported by the results from immunochemical staining of the SDS-polyacrylamide gel electrophoretogram of the purified isozymes and detergent-solubilized bovine liver microsomes transferred to the nitrocellulose sheet. Both cytochromes P-450 showed high catalytic activities toward (+)-benzphetamine and aminopyrine in reconstituted systems, indicating that both enzymes have a high turnover number for N-demethylation.

Postnatal development of constitutive forms of cytochrome p-450 in liver microsomes of male and female rats

In a previous paper (1), we reported on the purification of constitutive forms of cytochrome P-450, namely P-450-male and P-450-female, from liver microsomes of male and female rats, respectively. Immunochemical examinations of these hemoproteins showed that P-450-male and P-450-female were detectable specifically in respective liver microsomes of adult male and female rats. The synthesis of P-450-male was apparently dependent on testosterone, and that of P-450-female was dependent on estradiol. Thus, in this study we examined the postnatal development of P-450-male and P-450-female. We show herein that P-450-female is primarily synthesized before the occurrence of P-450-male in male rats. This and other results support the view that the synthesis of unknown pre-existing forms of cytochrome P-450 is depressed in association with the appearance of P-450-male and P-450-female during postnatal periods before sexual maturation.

Sex difference of cytochrome P-450 in the rat: Purification, characterization, and quantitation of constitutive forms of cytochrome P-450 from liver microsomes of male and female rats

One of each constitutive form of cytochrome P-450 from liver microsomes of adult male and female rats was purified essentially following the same method to an apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights estimated by the electrophoresis were 52,000 and 50,000 for forms of cytochrome P-450, P-450-male, and P-450-female, purified from male and female rats, respectively. In addition, the purified preparations of P-450-male and P-450-female showed properties different from each other with respect to spectral characteristics and catalytic activities. In Ouchterlony double diffusion plates, partially purified rabbit immunoglobulin G (IgG) raised against P-450-male and P-450-female showed very weak or no cross-reactivity with P-450-female and P-450-male, respectively. From these results, P-450-male was confirmed to be a form distinct from P-450-female. The anti- P-450-male and anti- P-450-female antibodies, which had been further purified by immunoadsorption, did not form any apparent precipitation bands with liver microsomes from untreated female and male rats, respectively. Supporting this, radial immunodiffusion analysis for P-450-male and P-450-female with an agarose gel impregnated with the rabbit antibodies showed that P-450-male and P-450-female appear in liver microsomes rather specifically depending on the sex hormones. Based on these results, sex differences in drug metabolism in the rat were confirmed as explicable, at least in part, by the presence of distinct forms of cytochrome P-450 in microsomes of male and female rats.

The effect of enzyme induction on the stereoselective metabolism of optically pure (-)1R,2R- and (+)1S,2S-dihydroxy-1,2-dihydrobenz-[a]anthracenes to vicinal 1,2-dihydrodiol 3,4-epoxides by rat liver microsomes.

The optically pure (-) and (+)trans-1,2-dihydroxy-1,2-dihydrobenz[a]anthracenes (BA trans-1,2-dihydrodiol) were obtained through the resolution of their diastereomeric di(-)menthoxyacetates by normal-phase h.p.l.c., followed with base-catalyzed hydrolysis. The (-)-BA trans-1,2-dihydrobiol has been determined to have 1R,2R absolute stereochemistry by exciton chirality method. Each of the enantiomeric and racemic BA trans-1,2-dihydrodiol was incubated with liver microsomes from untreated, phenobarbital (PB)-, and 3-methylcholanthrene (MC)-treated male Sprague-Dawley rats. The racemic and enantiomeric BA trans-1,2-dihydrodiols were each metabolized to two 1,2,3,4-tetrahydroxy-1,2,3,4-tetrahydrobenz[a]anthracenes (BA 1,2,3,4-tetrol) derived from the hydrolysis of BA trans-1,2-dihydrodiol anti-3,4-epoxide (the 3,4-epoxy oxygen is trans to the 1-hydroxyl group) and syn-3,4-epoxide (the 3,4-epoxy oxygen is cis to the 1-hydroxyl group), respectively. All the BA 1,2,3,4-tetrols were identified by comparing the reversed-phase h.p.l.c. retention times of tetrols and their vicinal acetonides with the hydrolysis products of the chemically synthesized BA trans-1,2-dihydrodiol anti-3,4-epoxide, BA trans-1,2-dihydrodiol syn-3,4-epoxide, BA trans-3,4-dihydrobiol anti-1,2-epoxide, and BA trans-3,4-dihydrodiol syn-1,2-epoxide, respectively, by their modes of forming vicinal acetonides, and by ultraviolet absorption and mass spectral analyses. From the metabolism of (-)-BA trans-1,2-dihydrodiol, a BA trans-1,2-dihydrodiol anti-3,4-epoxide was the major product formed by liver microsomes from MC-treated rats whereas a BA trans-1,2-dihydrodiol syn-3,4-epoxide was the major product formed by liver microsomes from either untreated or PB-treated rats. In contrast, BA trans-1,2-dihydrodiol syn-3,4-epoxide was the major product formed from the metabolism of (+)-BA trans-1,2-dihydrodiol by all three rat liver microsomal preparations. Liver microsomes from PB-treated rats were found to catalyze the metabolism of both the racemic and the enantiomeric BA trans-1,2-dihydrodiols at a rate higher than those by liver microsomes from untreated and MC-treated rats. All BA 1,2,3,4-tetrol metabolites were found to be optically active by circular dichroism spectral analysis. The results indicate that the 'bay-region' BA trans-1,2-dihydrodiol is metabolized by rat liver microsomes predominantly at the vicinal 3,4-double bond and that each enantiomeric BA trans-1,2-dihydrodiol is metabolized to a pair of diastereomeric BA trans-1,2-dihydrodiol 3,4-epoxides with varying degrees of stereoselectivity depending on the constitutive forms of cytochrome P-450 in the rat liver microsomal preparations.

Purification and characterization of two constitutive forms of rat liver microsomal cytochrome P-450.

Two constitutive forms of cytochrome P-450 isozymes, designated RLM3 and RLM5, have been purified from untreated rat liver microsomes. RLM3 and RLM5 have minimum molecular weights of 50,000 and 51,000, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Absolute oxidized spectra indicate that RLM3 is essentially all in the low spin state, whereas RLM5 contains some high spin component. The CO-reduced difference spectral maximum of RLM3 is at 449 nm and RLM5 is at 451 nm. RLM3 preferentially hydroxylates testosterone at 6 beta and 7 alpha positions and forms an even higher amount of an as yet unidentified polar metabolite, whereas RLM5 hydroxylates testosterone preferentially at 2 beta and 16 alpha positions, and exhibits 6 beta-hydroxylase activity similar to RLM3. RLM5 metabolizes aminopyrine, ethylmorphine, and benzphetamine all faster than does RLM3. When RLM3 and RLM5 were digested with proteolytic enzymes, peptide maps showed that these two cytochrome P-450 isozymes have different primary structures. Cyanogen bromide digestion breaks both isozymes into fragments too small to compare on polyacrylamide gels. Amino acid analyses indicate that RLM3 and RLM5 have very similar but not identical compositions. NH2-terminal residues were determined by Edman degradation and high pressure liquid chromatography separation of phenylthiohydantoin-derivatives. The first four residues of both enzymes are identical, NH2-Met-Asp-Pro-Val, but the fifth residue of RLM5 is -Leu-while that of RLM3 is -Val-. These sequences differ dramatically from all other P-450 isozymes reported to date.

Purification and properties of P-450 lm3b, a constitutive form of cytochrome P-450, from rabbit liver microsomes

This laboratory has previously reported the occurrence in rabbit liver microsomes of a non-inducible form of cytochrome P-450, designated P-450 lm 3b because of its electrophoretic mobility relative to that of phenobarbital-inducible P-450 lm 2 and 5,6-benzoflavone-inducible P-450 lm 4. In the present study, P-450 lm 3b was purified to electrophoretic homogeneity and a specific content of over 19 nmol per mg of protein by chromatographic procedures carried out in the presence of detergents. The isolated cytochrome has a minimal molecular weight of 52,000 and exhibits absorption maxima at 418, 537, and 571 nm in the oxidized state, 412 and 547 nm in the reduced state, and 451 and 555 nm as the CO complex. In a reconstituted system containing NADPH-cytochrome P-450 reductase and phosphatidylcholine, P-450 lm 3b has relatively high activity in the hydroxylation of testosterone in the 6β and 16α positions as well as significant activity toward a number of other substrates tested. The NADPH oxidase activity of P-450 lm 3b is less than half that of P-450 lm 2 and lm 4.