Pituitary regulation of sex-specific forms of cytochrome P-450 in liver microsomes of rats

Effects of hypophysectomy and treatment with testosterone or estradiol on the sex-specific forms of cytochrome P-450, P-450-male and P-450-female, were examined. The amounts of P-450-male as well as drug oxidation activities were decreased by hypophysectomy of male rats. In female rats, drug oxidation activities were increased by hypophysectomy, which was associated with the disappearance of P-450-female and the appearance of P-450-male. Treatment of hypophysectomized female rats with testosterone or estrodiol effected minor changes in the amounts of P-450-male.     

Effects of phenobarbital, 3-methylcholanthrene and polychlorinated biphenyls on sex-specific forms of cytochrome P-450 in liver microsomes of rats

The effects of treatment with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyls (PCB) on the amounts of sex-specific forms of cytochrome P-450, namely P-450-male and P-450-female, in male and female rats were studied. Although treatment with phenobarbital, 3-methylcholanthrene or PCB markedly increased the total amount of hepatic cytochrome P-450, P-450-male and P-450-female were rather decreased or not significantly changed. Thus, the percentages of P-450-male and P-450-female in the total cytochrome P-450 were decreased in liver microsomes from the treated rats. The increases in specific cytochrome P-450, such as P-448-H, P-448-L, and P-450I-c accounted for the increase in the total amount of cytochrome P-450 in the treated rats. The treatment with phenobarbital or PCB increased the activities of testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase and aminopyrine N-demethylase more markedly in female rats than in male rats. Similarly, the treatment with 3-methylcholanthrene increased benzo(a)pyrene hydroxylase more markedly in female rats. Therefore, the sex-differences in testosterone 16 alpha-hydroxylase, benzo(a)pyrene hydroxylase, and aminopyrine N-demethylase activities became smaller after the drug treatment. These results indicate that sex-specific P-450-male and P-450-female were unaffected, or even depressed by the agents in some cases.     

Effect of growth hormone and ectopic transplantation of pituitary gland on sex-specific forms of cytochrome P-450 and testosterone and drug oxidations in rat liver

The effects of growth hormone and ectopic transplantation of pituitary gland on the amounts of sex-specific cytochrome P-450, P-450-male and P-450-female, and the activities of testosterone and drug hydroxylases in male rat liver microsomes were studied. Hypophysectomy decreased the content of P-450-male, without changing the total cytochrome P-450 level. The continuous infusion of growth hormone into hypophysectomized rats and the transplantation of pituitary gland under the renal capsule caused a further decrease in P-450-male content and an expression of P-450-female. In contrast, the intermittent injection of growth hormone into hypophysectomized rats increased P-450-male content to the level seen in intact male rats. The activities of testosterone 2 alpha- and 16 alpha-, but not 6 beta-, 7 alpha-, or 15 alpha-hydroxylase, were changed in association with the level of P-450-male by these treatments. Anti-P-450-male immunoglobulin G inhibited testosterone 2 alpha- and 16 alpha-hydroxylations, but not 6 beta-, 7 alpha- or 15 alpha-hydroxylation. These results indicate that growth hormone regulates the expression of P-450-male responsible for testosterone 2 alpha- and 16 alpha-hydroxylations. The metabolism of 7-propoxycoumarin, benzo(a)pyrene and aminopyrine also changed with the content of P-450-male, although the correlation was less than that observed with testosterone 2 alpha- and 16 alpha-hydroxylation.     

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.     

Suppression of levels of phenobarbital-inducible rat liver cytochrome P-450 by pituitary hormone

The effect of pituitary factor on the constitutive and inducible levels of hepatic phenobarbital (PB)-inducible major cytochrome P-450, P-450b and P-450e, in male and female rat livers was studied by immunoblot analyses. Although only trace amounts (approximately 4 pmol/mg protein) of P-450b and P-450e were detected in untreated adult rats, hypophysectomy increased the contents of P-450b and P-450e 58- and 14-fold, respectively, in male rats and 118- and 30-fold, respectively, in female rats. The increases were also observed in treatment with dexamethasone, which suppressed the pituitary function. Treatment with PB increased more effectively the hepatic contents of P-450b and P-450e, but their contents were still 4-fold higher in the male than the female. Treatment of hypophysectomized female rats with PB increased the contents of P-450b and P-450e 4-fold higher than the contents in PB-treated nonhypophysectomized female rats. Consequently, the sex-related difference in their contents was reduced less than 1.4-fold in the hypophysectomized rats treated with PB. Similar results were also obtained from the quantitation of microsomal O-pentylresorufin O-depentylation and testosterone 16 beta-hydroxylation. Either intermittent injection or continuous infusion of human growth hormone, but not of ovine prolactin, into hypophysectomized male and female rats decreased the contents of both cytochromes. These results indicate that growth hormone acts as a repressive factor for the constitutive and inducible levels of P-450b and P-450e in a manner different from the regulation of P-450-male and P-450-female.     

Mutagenic activation of aflatoxin B1 by several forms of purified cytochrome P-450

Metabolic activation by several forms of purified cytochrome P-450 of aflatoxin B1 to a product(s) mutagenic to Salmonella typhimurium TA100 was examined. Of the 5 forms of cytochrome P-450 purified from liver microsomes of untreated and PCB-treated male rats, a constitutive form purified from untreated male rats, P-450-male, and a high-spin form of cytochrome P-450, P-448-H, from PCB-treated rats were highly active.     

Studies on in vitro antipyrine metabolism by 13C,15N double labeled method

The capacities of forms of cytochrome P-450 to oxidize antipyrine were compared. An isotope dilution gas chromatography/mass spectrometry/selected ion monitoring assay was developed to quantify the three main metabolites, norantipyrine, 3-hydroxymethylantipyrine and 4-hydroxyantipyrine. 13C,15N-Double labeled antipyrine was used as a substrate and the metabolites were analyzed as their trimethylsilyl derivatives. Among forms of cytochrome P-450 examined, a male-specific form of P-450, namely P-450-male, showed higher activity to form all the three metabolites. The other forms were responsible only for the formation of norantipyrine and 4-hydroxyantipyrine. The activities of liver microsomes from untreated male and female rats and rats treated with phenobarbital, 3-methylcholanthrene or polychlorinated biphenyl were expressed dependent on the activities of forms of cytochrome P-450 examined.     

A sex-specific form of cytochrome P-450 catalyzing propoxycoumarin O-depropylation and its identity with testosterone 6 beta-hydroxylase in untreated rat livers: reconstitution of the activity with microsomal lipids

Characteristics of a typical male-dominant reaction, dealkylation of n-propoxycoumarin, in rat livers were studied in relation to microsomal testosterone 6 beta-hydroxylase. The depropylation was more than 10-fold higher in the liver of male than female adult rats, but the sex-related difference was eliminated by neonatal castration. Hypophysectomy of adult male rats, which decreased the rates of male-specific P-450-male-dependent reactions, increased the depropylation of propoxycoumarin, while the rate was decreased by either intermittent injection or continuous infusion of human growth hormone to hypophysectomized rats. With regard to age-related difference, microsomal depropylation was detectable at neonate and reached a maximal level at 14 to 20 d of age, but was abruptly diminished only in female rats at puberty. These changes are in good agreement with those of testosterone 6 beta-hydroxylation and the content of a male-specific P-450(6)beta-1/PB-1. In reconstituted systems using extracted microsomal lipids, P-450(6)beta-1/PB-1 and P-450-male catalyzed the depropylation of propoxycoumarin. However, the microsomal depropylation was inhibited by antibodies which recognize P-450(6)beta-1/PB-1, but not P-450-male. These results indicate that microsomal depropylation of propoxycoumarin is catalyzed mainly by a male-specific P-450(6)beta-1/PB-1 in livers of untreated rats.     

Regulation of testosterone hydroxylation by rat liver microsomal cytochrome P-450

The pathways of testosterone oxidation catalyzed by purified and membrane-bound forms of rat liver microsomal cytochrome P-450 were examined with an HPLC system capable of resolving 14 potential hydroxylated metabolites of testosterone and androstenedione. Seven pathways of testosterone oxidation, namely the 2 alpha-, 2 beta-, 6 beta-, 15 beta-, 16 alpha-, and 18-hydroxylation of testosterone and 17-oxidation to androstenedione, were sexually differentiated in mature rats (male/female = 7-200 fold) but not in immature rats. Developmental changes in two cytochrome P-450 isozymes largely accounted for this sexual differentiation. The selective expression of cytochrome P-450h in mature male rats largely accounted for the male-specific, postpubertal increase in the rate of testosterone 2 alpha-, 16 alpha, and 17-oxidation, whereas the selective repression of cytochrome P-450p in female rats accounted for the female-specific, postpubertal decline in testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity. A variety of cytochrome P-450p inducers, when administered to mature female rats, markedly increased (up to 130-fold) the rate of testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylation. These four pathways of testosterone hydroxylation were catalyzed by partially purified cytochrome P-450p, and were selectively stimulated when liver microsomes from troleandomycin- or erythromycin estolate-induced rats were treated with potassium ferricyanide, which dissociates the complex between cytochrome P-450p and these macrolide antibiotics. Just as the testosterone 2 beta-, 6 beta-, 15 beta-, and 18-hydroxylase activity reflected the levels of cytochrome P-450p in rat liver microsomes, so testosterone 7 alpha-hydroxylase activity reflected the levels of cytochrome P-450a; 16 beta-hydroxylase activity the levels of cytochrome P-450b; and 2 alpha-hydroxylase activity the levels of cytochrome P-450h. It is concluded that the regio- and stereoselective hydroxylation of testosterone provides a functional basis to study simultaneously the regulation of several distinct isozymes of rat liver microsomal cytochrome P-450.     

Cytochrome P-450 in human liver microsomes: high-performance liquid chromatographic isolation of three forms and their characterization

Three forms of cytochrome P-450, designated as P-450-HM1, P-450-HM2, and P-450-HM3, were isolated from human liver microsomes using high-performance liquid chromatography (HPLC) techniques. Each purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the results of SDS-PAGE, the molecular weights of P-450-HM1, P-450-HM2, and P-450-HM3 were estimated to be 51,000, 54,000, and 52,000, respectively. The oxidized absolute spectra of these three forms of cytochrome P-450 showed Soret absorption peaks at around 417 nm, indicating that these forms were in the low spin state. In a reconstituted system, P-450-HM1 showed the highest catalytic activities of nifedipine and (S)- or (R)-nilvadipine oxidases. The same form showed higher activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. P-450-HM2 showed high N-demethylase activities for benz-phetamine and aminopyrine, and also showed the highest activity of testosterone 16 beta-hydroxylase among the three forms, while it did not show detectable activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. Anti-P-450-HM1 immunoglobulin G (IgG), but not anti-P-450-HM2 IgG, inhibited the activities of testosterone 6 beta-hydroxylase and nifedipine and nilvadipine oxidases in human liver microsomes. Anti-P-450-HM1 IgG was also inhibitory against progesterone 6 beta- and 16 alpha-hydroxylases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neonatal imprinting and hepatic cytochrome P-450 II. Partial purification of a sex-dependent and neonatally imprinted form(s) of cytochrome P-450

A new form of cytochrome P-450 was partially purified from hepatic microsomes of neonatally imprinted rats (adult male and adult male castrated at four weeks of age). This new form of cytochrome P-450 appears to have an apparent molecular weight of approximately 50,000 daltons as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. It appears that this form of cytochrome P-450 is either absent or present in low concentrations in cytochrome P-450 preparations isolated from neonatally nonimprinted rats (adult female and adult male castrated at birth). Reconstitution of testosterone hydroxylase and benzphetamine N-demethylase activities of this partially purified cytochrome P-450 revealed that the presence of testosterone 16α-hydroxylase activity, an imprintable microsomal enzyme, was in parallel with the imprinting status of the animals; a significantly higher activity was detected in the neonatally imprinted than that of the nonimprinted animals. This was in contrast to the nonimprintable benzphetamine N-demethylase, testosterone 7α-and 6β-hydroxylase activities which exhibited no correlation with the imprinting status of the animals. We have prepared antisera from rabbits using the partially purified cytochrome P-450 preparations from adult male rats as antigens. These antisera inhibited microsomal testosterone 16α- and 7α-hydroxylase activities in a concentration-dependent manner, without impairing 6β-hydroxylase activity. These data suggest that the partially purified cytochrome P-450 from adult male rats consists of both imprintable (16α-) and nonimprintable (7α-) testosterone hydroxylase activities. The antisera formed immunoprecipitant lines in the Ouchterlony double diffusion plates with partially purified cytochrome P-450 from both neonatally imprinted and nonimprinted adult rats. The immunoprecipitant lines, as stained by coomassie blue, suggest the homology of the cytochrome P-450 preparations from neonatally imprinted and nonimprinted rats. Immunoabsorption of the antisera against neonatally nonimprinted, partially purified cytochrome P-450 completely removed the immunoprecipitant lines without appreciably impairing the inhibitory effects of antisera on the microsomal testosterone 16α-and 7α-hydroxylase activities. In contrast, immunoabsorption of the antisera against partially purified cytochrome P-450 from adult male rats (imprinted) abolished completely both the immunoprecipitant lines and the inhibition on microsomal testosterone hydroxylation reaction (16α and 7α). The inhibitory actin of antisera on testosterone hydroxyulation was also abolished upon boiling the antisera at 100°C for 5 minutes. The biochemical and immunochemical data in this study suggest that the neonatally imprintable form or forms of hepatic microsomal cytochrome P-450 accounts for a small fraction of the bulk of total cytochrome P-450. However, the existence of this form of cytochrome P-450 is regulated by gonadal hormones during the neonatal period and accounts for the major imprintable sex difference in drug and steroid metabolism in adulthood.     

Purification of human liver cytochrome P-450 catalyzing testosterone 6 beta-hydroxylation

Two forms of cytochrome P-450 (P-450 human-1 and P-450 human-2) have been purified from human liver microsomes to electrophoretic homogeneity. P-450 human-1 and P-450 human-2 differ in their apparent molecular weights (52,000 and 56,000, respectively) and Soret peak maxima in the CO-binding reduced difference spectrum (447.6 and 450.3 nm, respectively). In the reconstituted system using rat liver NADPH-cytochrome c (P-450) reductase, P-450 human-2 more effectively oxidized benzo(a)pyrene (80-fold), ethylmorphine (2-fold), and 7-ethoxycoumarin (2-fold) than did P-450 human-1. However, P-450 human-1 showed higher testosterone 6 beta-hydroxylase activity, and the activity was markedly increased by the inclusion of cytochrome b5 or spermine in the reconstituted system. Antibodies raised against P-450 human-1 inhibited more than 80% of microsomal testosterone 6 beta-hydroxylase activity in human liver. Immunoblotting analysis using anti-P-450 human-1 IgG revealed a single immuno-staining band near Mr 52,000 in all human liver samples examined. The amount of immunochemically determined P-450 human-1 varied in parallel with the testosterone 6 beta-hydroxylase activity in human liver. These results indicate that P-450 human-1 is a major form of cytochrome P-450 responsible for microsomal testosterone 6 beta-hydroxylation. Thus, this paper is the first report on human cytochrome P-450 responsible for testosterone 6 beta-hydroxylation, which is the major hydroxylation pathway in human liver microsomes.     

Feminization of rat hepatic P-450 expression by cisplatin. Evidence for perturbations in the hormonal regulation of steroid-metabolizing enzymes

The biochemical basis for the complex effects of the anti-cancer drug cisplatin on hepatic cytochrome P-450 activity was studied in adult male rat liver using P-450 form-specific steroid hydroxylase assays and antibody probes. Cisplatin treatment of adult male rats resulted in a marked and prolonged feminization of the pattern of P-450 enzymes expressed in hepatic tissue. The adult male-specific cytochrome P-450 forms designated P-450 2c (P-450 gene IIC11), P-450 2a (gene IIIA2), and P-450 RLM2 were decreased by 70-90% after 7-14 days, with parallel decreases in their respectively associated microsomal steroid hydroxylase activities. Concomitantly, hepatic levels of the female-predominant enzymes P-450 3 (gene IIA1) and P-450j (gene IIE1) were elevated approximately 2-4-fold. The female-specific microsomal enzyme androstenedione 5 alpha-reductase was induced approximately 20-fold by cisplatin; however, no elevation of the female-specific P-450 2d was detected. The underlying hormonal basis for these effects of cisplatin was then examined. Serum testosterone levels were found to be depleted by cisplatin in a time- and dose-dependent manner which correlated with the observed changes in these hepatic enzymes. Furthermore, castration of adult rats altered the profile of these enzymes in a manner which resembled that observed with cisplatin treatment, suggesting that androgen depletion was the primary cause for the observed feminization of hepatic enzyme expression. Consistent with this possibility, the synthetic androgen methyltrienolone effectively blocked the changes in hepatic enzyme expression induced by cisplatin. Moreover, hepatic enzyme feminization was significantly reversed by chorionic gonadotropin, which fully restored serum testosterone levels in the cisplatin-treated rat. Luteinizing hormone-releasing hormone challenge experiments demonstrated that the responsiveness of the pituitary to this hypothalamic regulator of testicular androgen production was unimpaired by cisplatin treatment, indicating that hypothalamic production or secretion of luteinizing hormone-releasing hormone may be deficient in the cisplatin-treated animals. These studies establish that the effects of cisplatin on hepatic P-450 enzyme expression result from its interruption of the hypothalamic-pituitary stimulation of testicular androgen production and that this, in turn, leads to a depletion of circulating androgens required for maintenance of normal P-450 enzyme expression in adult male rats.     

Increased activity of testosterone hydroxylases in liver microsomes of diabetic rats treated with insulin

Liver microsomes from alloxan diabetic rats displayed decreased activity to hydroxylate testosterone only at the 2-alpha and 6-beta positions. Diabetic insulin-treated rats showed higher hydroxylase activities than diabetic and control rats in the formation of all testosterone metabolites analyzed. The sodium dodecylsulfate electrophoretic profile of liver microsomal proteins from each group of rats exhibited distinct increases as well as decreases in the cytochrome P-450 region. Stimulation of testosterone metabolism by insulin may be associated with a higher synthesis of certain cytochrome P-450 isozymes.     

Regulation of steroidogenesis in rat Leydig cells in culture: effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin

Rat Leydig cells in primary culture were used as a model system to investigate the effects of human chorionic gonadotropin (hCG) and dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and the iron-sulfur protein, adrenodoxin. Leydig cells isolated from the testes of mature rats were placed in monolayer culture in the absence of stimulatory factors for 8 days. HCG (10 mIU/ml) or Bt2cAMP (1 mM) were then added to some of the cultures and the incubations were continued for up to 48 h. Testosterone production was increased markedly in cells incubated with hCG or Bt2cAMP. A significant accumulation of pregnenolone in the medium of cells treated with Bt2cAMP was also observed. Both hCG and Bt2cAMP increased the rates of synthesis of cytochrome P-450scc and adrenodoxin. In hCG-treated cells the apparent rate of synthesis of cytochrome P-450scc was increased 13-fold over that of controls after 48 h of incubation; the rate of adrenodoxin synthesis was increased 4-fold by hCG treatment. In Bt2cAMP-treated cells the rate of synthesis of cytochrome P-450scc was 37-fold greater than that of control cells after 48 h of incubation; adrenodoxin synthesis was increased 36-fold over controls. In hCG- and Bt2cAMP-treated cells, the concentration of immunoreactive cytochrome P-450scc and adrenodoxin increased with increasing time of incubation, and were correlated with the stimulatory effects of these agents on cytochrome P-450scc activity and on total steroid production. The results of this study are indicative that the maintenance by LH/hCG of elevated levels of testosterone synthesis by the Leydig cell is mediated, in part, by induction of the synthesis of cytochrome P-450scc and its associated protein, adrenodoxin. Since Bt2cAMP had effects similar to those observed with hCG, it is suggested that the stimulatory effects of hCG on the synthesis of cytochrome P-450scc and adrenodoxin are mediated by increased cyclic AMP formation.     

Tissue-specific regulation of cytochrome P-450 dependent testosterone 15 alpha-hydroxylase

Testosterone 15 alpha-hydroxylase activity in kidney microsomes is higher in male mice than in female mice, while in the liver the activity is higher in females than in males. Cytochrome P-450 15 alpha, a specific form of cytochrome P-450 having testosterone 15 alpha-hydroxylase activity, accounts for virtually all of the testosterone 15 alpha-hydroxylase activity in female kidney microsomes, while other isozymes of testosterone 15 alpha-hydroxylase are present in male kidney microsomes. In female kidney, P-450 15 alpha expression is regulated by a single sex-dependent locus, called Rsh for "regulation of steroid hydroxylase." The higher level of P-450 15 alpha expression in male kidneys is dependent on androgens. Of all mice strains, 129/J seems to be the least dependent on androgens to maintain a high expression of P-450 15 alpha in male kidneys. Castration of male mice lowers kidney levels of P-450 15 alpha but in the liver, P-450 15 alpha levels rise after castration. This reciprocal regulation of P-450 15 alpha genes in liver and kidney was investigated by isolating cDNA clones encoding P-450 15 alpha from liver and kidney cDNA libraries. Two highly homologous cDNA clones encoding P-450 15 alpha designated type I and type II were identified, and levels of type I and type II mRNA in liver and kidney were determined by differential restriction mapping of double-stranded cDNA prepared from mRNA from these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleotide sequence of a human liver cytochrome P-450 related to the rat male specific form

P-450 human-2 is a human cytochrome P-450 that is immunochemically related to a constitutive male-specific cytochrome P-450 (P-450-male) and the phenobarbital-inducible P-450b/e in rat liver. By screening a human liver cDNA library in bacteriophage lambda gt11, we isolated a clone with an insert length of 1,847 bases (pHY13). The clone was sequenced and shown to code for a protein of 487 amino acids. The N-terminal 11-amino-acid sequence was in agreement with the protein sequence of P-450 human-2. The nucleotide sequence of pHY13 showed less than 50% similarity with those of human cytochrome P-450s, pHP-450(1), HLp, P-450NF, P1-450 4, and P3(450), but the nucleotide sequence of pHY13 is 80% similar to the reported sequence of rat cytochrome P-450, P-450(M-1). In addition, the coding sequence of pHY13 showed close similarity to that of MP-8, which was recently reported as the sequence corresponding to human cytochrome P-450MP, although no apparent similarity was observed in their 3' non-coding sequences except for the first 75 bases and the expected length of the complete sequences. These results, together with the immunochemical data, indicate that P-450 human-2 is closely related, but not identical, to P-450MP, and may belong to the category of developmentally regulated constitutive cytochrome P-450s.     

Theophylline metabolism by rat liver microsomal monooxygenases

Theophylline metabolism has been studied in a reconstituted monooxygenase system with purified forms of cytochrome P-450: P-450a, P-450b, P-450d and P-450k as well as in liver microsomes of control and 3-methylcholanthrene-induced rats. Cytochrome P-450 isoforms, P-450a and P-450b, had no effect on theophylline metabolism, whereas forms P-450d and P-450k induced the synthesis of 1.3-dimethyluric acid (1.3-DMA) at the rates of 900 and 330 pmol/min/nmol of protein, respectively. The catalytic activity of these isoforms was fully inhibited by homologous monospecific antibodies. P-450c catalyzed the formation of a nonidentified metabolite. In microsomes of control animals antibodies specifically directed to cytochrome P-450k suppressed the rate of 1.3-DMA synthesis by 73%, whereas antibodies specifically raised against P-450c+d--by 11%. In microsomes of methylcholanthrene-induced animals the rate of 1.3-DMA synthesis was increased two-fold. This activity was inhibited by 61% by antibodies to cytochrome P-450k and by 18% by anti-P-450c+d antibodies.     

Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction

A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.     

Cytochrome P-450 isozymes from the marine teleost Stenotomus chrysops: their roles in steroid hydroxylation and the influence of cytochrome b5

Two new cytochrome P-450 forms were purified from liver microsomes of the marine fish Stenotomus chrysops (scup). Cytochrome P-450A (Mr = 52.5K) had a CO-ligated, reduced difference spectrum lambda max at 447.5 nm, and reconstituted modest benzo[a]pyrene hydroxylase activity (0.16 nmol/min/nmol P-450) and ethoxycoumarin O-deethylase activity (0.42 nmol/min/nmol P-450). Cytochrome P-450A reconstituted under optimal conditions catalyzed hydroxylation of testosterone almost exclusively at the 6 beta position (0.8 nmol/min/nmol P-450) and also catalyzed 2-hydroxylation of estradiol. Cytochrome P-450A is active toward steroid substrates and we propose that it is a major contributor to microsomal testosterone 6 beta-hydroxylase activity. Cytochrome P-450A had a requirement for conspecific (scup) NADPH-cytochrome P-450 reductase and all reconstituted activities examined were stimulated by the addition of purified scup cytochrome b5. Cytochrome P-450B (Mr = 45.9K) had a CO-ligated, reduced difference spectrum lambda max at 449.5 nm and displayed low rates of reconstituted catalytic activities. However, cytochrome P-450B oxidized testosterone at several different sites including the 15 alpha position (0.07 nmol/min/nmol P-450). Both cytochromes P-450A and P-450B were distinct from the major benzo[a]pyrene hydroxylating form, cytochrome P-450E, by the criteria of spectroscopic properties, substrate profiles, minimum molecular weights on NaDodSO4-polyacrylamide gels, peptide mapping and lack of cross-reaction with antibody raised against cytochrome P-450E. Cytochrome P-450E shares epitopes with rat cytochrome P-450c indicating it is the equivalent enzyme, but possible homology between scup cytochromes P-450A or P-450B and known P-450 isozymes in other vertebrate groups is uncertain, although functional analogs exist.