Inhibition of cholesterol 7 alpha-hydroxylase by an antibody to a male-specific form of cytochrome P-450 from subfamily P450IIC.

The absence of antibodies to cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), the rate-determining enzyme for bile acid synthesis, has significantly compromised studies on this protein. Nine antibodies raised against proteins from the cytochrome P-450 gene families (P450I, P450IIA, P450IIB, P450IIC and P450III) were tested as inhibitors of 7 alpha-hydroxylase activity. An antibody raised against a male-predominant P-450 (PB2a, P450h) from the P450IIC gene subfamily was an effective inhibitor of activity in liver microsomal fractions from rat, mouse and hamster. The inhibition could be reversed by the addition of PB2a antigen, indicating structural similarity between cholesterol 7 alpha-hydroxylase and proteins within the P450IIC subfamily. Western blot analysis of hepatic microsomal fractions with the PB2a antibody gave three bands, two of which, like cholesterol 7 alpha-hydroxylase, did not inhibit sexual dimorphism. The intensity of one of the bands (apparent Mr 54,000) correlated with changes observed in activity due to diet [Spearman correlation of 0.800 (P less than 0.01)]. These findings suggest that cholesterol 7 alpha-hydroxylase is a form of P-450 which shares structural similarity with cytochromes P-450 in the P450IIC gene subfamily and that its feedback regulation by bile acid involves protein induction rather than simply post-translational modification.     

The cDNA and protein sequence of a phenobarbital-induced chicken cytochrome P-450

Several cDNA clones complementary to a chicken phenobarbital-inducible cytochrome P-450 have been isolated and sequenced, representing the first non-mammalian eukaryotic cytochrome P-450 sequence to be analyzed. The cDNA clones hybridized to two mRNAs of 3.5 and 2.5 kilobases in length, but further analysis indicated that the clones were derived from the larger mRNA. The sequence contains a 5'-noncoding region of 39 nucleotides and an open reading frame of 1473 nucleotides. The remainder of the sequence is due to the 3'-noncoding region and poly(A) tail. The open reading frame encodes a protein of 491 amino acids with a molecular weight of 56,196. The chicken cytochrome P-450 shows an overall homology of 45-54% compared with the mammalian phenobarbital-induced cytochrome P-450s. The degree of homology is not uniform, with some short regions showing much greater levels of sequence conservation. In particular, the chicken cytochrome P-450 contains the conserved cysteinyl domain near the carboxyl terminus, found in all cytochrome P-450s and which is thought to be involved in heme binding. Using the chicken sequence, a more accurate estimate of the evolutionary rates of cytochrome P-450s has been made. It is suggested that the phenobarbital-, 3-methylcholanthrene, and pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450 gene families diverged from a common ancestral gene 600 million years ago. Furthermore the phenobarbital-inducible gene apparently underwent gene duplication events at about the time of the divergence of the chicken and mammalian lineages. The results imply that most mammals should have at least four rather distantly related phenobarbital-inducible gene subfamilies.     

Gene structure of a major form of phenobarbital-inducible cytochrome P-450 in rat liver

The gene structure of cytochrome P-450b, a major form of phenobarbital-inducible cytochrome P-450 in rat livers was elucidated by sequence analysis of the cloned genomic DNAs and was compared with the previously determined gene structures of cytochrome P-450e, a minor form of phenobarbital-inducible cytochrome P-450 and two forms of 3-methylcholanthrene-inducible cytochrome P-450 (P-450c and -d). The gene for cytochrome P-450b is 23 kilobase pairs (kb) long and is separated into 9 exons by 8 intervening sequences. This gene structure is very similar to that of cytochrome P-450e except for the first intron, the first intron being much longer in cytochrome P-450b gene (approximately 12 kb) than in cytochrome P-450e gene (3.2 kb), but differs greatly from the gene structures of two 3-methylcholanthrene-inducible cytochrome P-450s as pointed out previously (Sogawa, K., Gotoh, O., Kawajiri, K. & Fujii-Kuriyama, Y. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5066-5070). The nucleotide sequences in all 9 exons and their flanking regions in introns show very close homology between the two phenobarbital-inducible cytochrome P-450 genes. Forty base substitutions are found in approximately 1900 nucleotides of all exonic sequences, and 15 of them result in 14 amino acid replacements. These base substitutions occur in relatively limited regions of the gene sequences. Most of them are found in exons 6, 7, 8, and 9, most frequently in exon 7 as described previously (Mizukami, Y., Sogawa, K., Suwa, Y., Muramatsu, M. & Fujii-Kuriyama, Y. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3958-3962). The close sequence homology between the two phenobarbital-inducible cytochrome P-450 genes is also found to extend to the promoter region with one notable exception. The simple repeated sequences of (CA)n which is present at -254 position in cytochrome P-450e gene is also observed at the equivalent position in cytochrome P-450b gene, but the repetitiveness is greatly reduced in cytochrome P-450b gene ((CA)5 for P-450b versus (CA)19 for P-450e), and this may somehow be related to the difference in the level of cytochrome P-450b and P-450e in the inductive phase of phenobarbital administration.     

Polyphosphoinositide activation of cholesterol side chain cleavage with purified cytochrome P-450scc

Highly purified beef adrenal cytochrome P-450 specific for cholesterol side chain cleavage (P-450-scc) has been reconstituted with sonicated vesicles containing cholesterol and either dimyristoyl phosphatidylcholine (DMPC) or dioleoyl phosphatidylcholine (DOPC). When cholesterol was present in DMPC vesicles at 1:15 molar ratio, cardiolipin and L-alpha-phosphatidylinositol 4-monophosphate (DPI) increased side chain cleavage by at least 5-fold (0.7 min-1-3.5 min-1). In DOPC vesicles, a smaller increase was observed (2.8 min-1-5.0 min-1). Activator phospholipids increased the rate of transference of cholesterol both to and from the cytochrome when, respectively, cholesterol-free P-450scc and cholesterol-P-450scc complex are combined with either DMPC or DOPC vesicles. Transfer of cholesterol to and from cytochrome P-450 occurred with similar first order rate constants and was also independent of the concentrations of cholesterol vesicles and P-450. It is suggested that transfer in both directions is limited by the rate of insertion of P-450scc into the membrane. Phospholipid stimulatory effects for both cholesterol transfer and for activation of side chain cleavage occurred with the same ranking, even though cholesterol transfer, following reconstitution, was 5-10 times slower than the turnover of side chain cleavage. DPI increased Vmax for side chain cleavage in both DMPC and DOPC vesicles to the same rate (12 min-1) without effect on the Km for cholesterol, while cardiolipin both produced a similar increase in Vmax and decreased Km (cholesterol). This activation by DPI is attributed to more favorable incorporation of P-450scc in these membranes and is consistent with previously reported effects of acidic phospholipids on other mitochondrial proteins.     

Cytochrome P-450 of bovine adrenal mitochondria. Ligand binding to two forms resolved by EPR spectroscopy.

The binding of cholest-5-ene-3beta,20alpha-diol (20alpha-hydroxycholesterol), 11-deoxycorticosterone, and aminoglutethimide to cytochrome P-450 in bovine adrenal mitochondria was measured by changes in optical spectra at room temperature and by EPR spectra at 14 K. The two methods provided nearly identical quantitation of these interactions with cytochrome P-450. Two distinct high spin forms of cytochrome P-450 were revealed by EPR spectra. The predominant high spin species (g = 8.2) was decreased by addition of 20alpha-hydroxycholesterol and elevated pH but was increased by addition of cholesterol. The minor high spin species (g = 8.1) was incrreased by addition of deoxycorticosterone but decreased by low concentrations of metyrapone. The two forms were evidently not in equilibrium and have been assigned to distinct forms of cytochrome P-450 involved in, respectively, cholesterol side chain cleavage (P-450scc) and steroid 11beta hydroxylation (P-450(11)beta). The high spin states are derived from complexes of these P-450 cytochromes with endogenous substrates, which are, respectively, cholesterol and deoxycorticoids. A high to low spin transition was observed when these complexes were turned over by initiating hydroxylation with malate. The contributions of cytochromes P-450(11)beta and P-450scc to the low spin spectrum were also resolved by similar means. At least 20% of P-450scc is in the low spin state while about 90% of P-450(11)beta is low spin in isolated beef adrenal mitochondria. Low spin complexes of cytochrome P-450scc with 20alpha-hydroxycholesterol and 3beta-hydroxypregn-5-ene-20-one (pregnenolone) gave distinct EPR spectra. Aminoglutethimide interacted with the total cytochrome P-450 content of the bovine adrenal mitochondria forming low spin complexes. Both optical and EPR data indicated binding to two forms of cytochrome P-450. These results suggest a detailed correlation between the spin state and absorbance changes seen at room temperature, illustrate that EPR allows the distinction of two principal forms of P-450, and suggest that there is no appreciable change in the spin state of either cytochrome between 14 K and 300 K.     

Cytochrome P-450 of adrenal mitochondria. In vitro and in vivo changes in spin states.

Steroid-induced difference spectra have been used to examine the combination of cholesterol with adrenal mitochondrial cytochrome P-450 which participates in cholesterol side chain cleavage (P-450scc) and the depletion of cholesterol from the cytochrome which results from turnover of the enzyme system. Type I difference spectra-induced by cholest-5-ene-3beta, 25-diol (25-hydroxycholesterol) and cholest-5-ene-3beta, 20 alpha, 22R-triol (20alpha, 22R dihydroxycholesterol) have been used to quantitate binding of cholesterol to two sites (I and II) on cytochrome P-450scc. The action of adrenocorticotropic hormone (ACTH) in vivo and the action of calcium or phosphate ions on isolated mitochondria stimulate the combination of cholesterol with site I but not site II. Cholesterol derived from lecithin-cholesterol micelles, however, binds to both sites. Malate-induced cholesterol depletion occurred at a comparable rate to the transfer of cholesterol from lecithin-cholesterol micelles. However, a residual proportion of cholesterol-cytochrome P-450scc complexes remained, even after 10 min of exposure to malate, and was of similar magnitude in mitochondria from both cycloheximide-treated and stressed rats. It is suggested that this reflects a less reactive form of cholesterol-cytochrome complex. Steroid-induced difference spectra indicate that sites I and II on cytochrome P-450scc are similarly depleted after metabolism of mitochondrial cholesterol in vitro and after inhibition of the action of ACTH in vivo. Anaerobiosis of adrenal cells after excision of the accumulation of cholesterol at cytochrome P-450cc. When anaerobiosis was prevented, cytochrome P-450scc in the freshly isolated mitochondria was apparently essentially free of complexed cholesterol, irrespective of the extent of ACTH action. For 30 min after suspension of the mitochondria in 0.25 M sucrose at 4 degrees, cholesterol combines with cytochrome P-450scc. The extent of this process was not affected by the presence of cycloheximide during ether stress treatment of the rats. It is concluded that there are at least two pools of mitochondrial cholesterol with access to cytochrome P-450scc but that ACTH stimulates only the pool which most readily interacts with the cytochrome.     

Effects of two oral antimycotics, ketoconazole and fluconazole, upon steroidogenesis in rat adrenal cells in vitro

Rat adrenal cells were incubated with various concentrations of two orally active azole antimycotics in order to evaluate the effects on steroidogenesis. The first compound was ketoconazole, a well-known inhibitor not only of fungal cytochrome P-450 but at higher concentrations also of mammalian cytochrome P-450 dependent enzymes. The second was fluconazole, a newly developed oral antimycotic with a triazole structure, which likewise inhibits fungal cytochrome P-450. The influence of both drugs on mammalian cytochrome P-450 dependent enzymes was investigated in this study. Ketoconazole inhibited ACTH-stimulated corticosterone (IC50 = 0.9 microM) and aldosterone secretion (IC50 = 1.4 microM) and enhanced 11-deoxycorticosterone output at low concentrations but reduced it at higher concentrations. Radiotracer experiments with [3H]pregnenolone or [3H]11-deoxycorticosterone as exogenous substrates revealed a 50% inhibition of the oxidative substrate metabolism at about 1 microM ketoconazole. These effects could also be observed with fluconazole but occurred at concentrations approximately two orders of magnitude higher as compared to ketoconazole. We conclude that fluconazole has a much higher selectivity for fungal cytochrome P-450 than ketoconazole. The order of sensitivity of the cytochrome P-450 dependent enzymes of rat adrenal steroidogenesis to ketoconazole was the 11 beta/18-hydroxylase, the cholesterol side chain cleavage enzyme and the 21-hydroxylase with decreasing sensitivities.     

Studies on the genetic regulation of cytochrome P-450 production in Saccharomyces cerevisiae

An initial survey of 18 haploid strains of Saccharomyces cerevisiae revealed that only 3 of these strains could produce a detectable level of cytochrome P-450. A cross between a cytochrome P-450 producing strain of S. cerevisiae (B/B) and a non-producing strain (D22) gave a diploid which was a non-producer and a 2:2 segregation of producers to non-producers in meiotic tetrads. Of the two producers in each tetrad, one produced a higher level of cytochrome P-450 than the other. We deduce that cytochrome P-450 production in S. cerevisiae is regulated by a single nuclear gene and that a modifier gene is also involved which can enhance the amount of cytochrome P-450 synthesized. Benzo(a)pyrene (an inducer of P-450 in yeast) had no effect on the action of the regulatory gene.     

Differences in the cytochrome P-450 isoenzymes involved in the 2-hydroxylation of oestradiol and 17 alpha-ethinyloestradiol. Relative activities of rat and human liver enzymes.

The metabolism of oestradiol and 17 alpha-ethinyloestradiol to their 2-hydroxy derivatives is an important determinant in their biological effects. In this work, we have investigated which rat or human cytochrome P-450 isoenzymes are involved in catalysing these reactions. Oestradiol 2-hydroxylation was catalysed by a wide variety of rat cytochrome P-450s from gene families P450IA, P450IIB, P450IIC and P450IIIA. Interestingly, 17 alpha-ethinyloestradiol, which only differs structurally from oestradiol at a position distant from the site of oxidation, was metabolized predominantly by members of the P450IIC gene subfamily. In order to establish which enzymes are responsible for the oxidation of these substrates in man, antibodies to rat liver cytochrome P-450 isoenzymes were used to inhibit these reactions in a panel of human liver microsomal fractions. Also, possible correlations between the proteins recognized by the antibodies and the 2-hydroxylation rate were determined. These experiments provide evidence that 2-hydroxylation of 17 alpha-ethinyloestradiol in man is catalysed by cytochromes from the P450IIC, P450IIE and P450IIIA gene families. In contrast, the major proteins involved in oestradiol metabolism are from the P450IA gene family, although members of the P450IIC and P450IIE gene families may also play a role. These data demonstrate that the differences in the capacity of rat P-450s to metabolize these substrates are also present in the comparable enzymes involved in man, and that a variety of factors will determine the rate of disposition of these compounds in man.     

Common characteristics of the cytochrome P-450 system involved in 18- and 11 beta-hydroxylation of deoxycorticosterone in rat adrenals.

18- and 11beta-Hydroxylation of deoxycorticosterone and side chain cleavage of cholesterol were studied in mitochondria and submitochondrial reconstituted systems prepared from rat and bovine adrenals. A mass fragmentographic technique was used that allows determination of hydroxylation of both exogenous and endogenous cholesterol. The following results were obtained. (1) Treatment of rats with excess potassium chloride in drinking fluid increased mitochondrial cytochrome P-450 as well as 18- and 11beta-hydroxylase activity in the adrenals. Cholesterol side chain cleavage was not affected. In the presence of excess adrenodoxin and adrenodoxin reductase, cytochrome P-450 isolated from potassium chloride-treated rats had higher 18- and 11beta-hydroxylase activity per nmol than cytochrome P-450 isolated from control rats. The stimulatory effects on 18- and 11beta-hydroxylation were of similar magnitude. (2) Long-term treatment with ACTH increased cholesterol side chain cleavage in the adrenals but had no effect on 18- and 11beta-hydroxylase activity. The amount of cytochrome P-450 in the adrenals was not affected by the treatment. It was shown with isolated mitochondrial cytochrome P-450 in the presence of excess adrenodoxin and adrenodoxin reductase that the effect of ACTH was due to increase of side chain cleavage activity per nmol cytochrome P-450. Side chain cleavage of exogenous cholesterol was affected more than that of endogenous cholesterol. (3) Gel chromatography of soluble cytochrome P-450 prepared from rat and bovine adrenal mitochondria yielded chromatographic fractions having either a high 18- and 11beta-hydroxylase activity and a low cholesterol side chain cleavage activity or the reverse. The ratio between 18- and 11beta-hydroxylase activity was approximately constant, provided the origin of cytochrome P-450 was the same. (4) Addition of progesterone to incubations of deoxycorticosterone with soluble or insoluble rat adrenal cytochrome P-450 competitively inhibited 18- and 11beta-hydroxylation of deoxycorticosterone to the same degree. Addition of deoxycorticosterone competitively inhibited 11beta-hydroxylation of progesterone with the same system. Progesterone was not 18-hydroxylated by the system. From the results obtained, it is concluded that 18- and 11beta-hydroxylation have similar properties and that the binding site for deoxycorticosterone is similar or identical in the two hydroxylations. The possibility that the same specific type of cytochrome P-450 is responsible for both 18- and 11beta-hydroxylation of deoxycorticosterone is discussed.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

Artifacts in isoelectric focusing of the microsomal enzymes cytochrome P-450 and NADPH-cytochrome P-450 reductase.

Highly-purified rat liver microsomal cytochrome P-450 and NADPH-cytochrome P-450 reductase (NADPH-ferricytochrome oxidoreductase, EC 1.6.2.4) preparations gave rise to a large number of bands under a variety of isoelectric focusing conditions, as observed after staining for either zymogen or protein. The binding patterns were not independent of sample concentration and position of application, and eluted bands did not refocus as expected. The artifactual heterogeneity is attributed to strong protein-protein interactions and perhaps to complexation of proteins with carrier ampholytes. These findings suggest caution in using isoelectric focusing to resolve mixtures of membrane proteins.     

Use of cytochrome P-450scc to measure cholesterol-lipid interactions

The interaction of cholesterol with phospholipids has been studied with a variety of techniques; however, the possible consequences of such interactions in vivo have not been demonstrated. In this study, the cholesterol-dependent absorbance spectrum of cytochrome P-450scc was used to monitor cholesterol availability in both micellar and vesicular environments. By use of this approach, in conjunction with titration of putative cholesterol binding species, a tight, approximately equimolar complex of cholesterol and digitonin was demonstrated. Sphingomyelin (SM) (both the synthetic N-palmitoyl and bovine brain forms) gave sigmoidal titration curves, suggesting a cooperative interaction between this lipid and cholesterol. The interaction of bovine brain glycerolipids and cholesterol was weaker than that of SM and showed no cooperativity. The importance of the phospholipid head group in these interactions was established by the differences in the ability of synthetic 1-palmitoyl-2-oleoylphosphatidylcholine, -phosphatidylethanolamine, and -phosphatidylserine to affect cholesterol availability. Comparison of these results with those of the bovine brain phospholipids indicates that the acyl chain composition of these molecules is also important to these interactions. Titrations of SM in phospholipid vesicles containing cytochrome P-450scc and different types of phosphatidylcholine established that the SM-cholesterol interactions also occur in a bilayer membrane. This study demonstrates that the association of cholesterol with cytochrome P-450scc is inhibited by concentrations of SM commonly found in biological membranes. Therefore, such cholesterol-lipid interactions can potentially affect the function of membrane enzymes.     

Ferredoxins from two sulfonylurea herbicide monooxygenase systems in Streptomyces griseolus.

We have purified and characterized two ferredoxins, designated Fd-1 and Fd-2, from the soluble protein fraction of sulfonylurea herbicide induced Streptomyces griseolus. These cells have previously been shown to contain two inducible cytochromes P-450, P-450SU1 (CYP105A1) and P-450SU2 (CYP105B1), responsible for herbicide metabolism [O'Keefe, D. P., Romesser, J. A., & Leto, K. J. (1988) Arch. Microbiol. 149, 406-412]. Although Fd-2 is more effective, either ferredoxin can restore sulfonylurea monooxygenase activity to an aerobic mixture of NADPH, spinach ferredoxin:NADP oxidoreductase, purified cytochrome P-450SU1, and herbicide substrate. The gene for Fd-1 is located in the genome just downstream of the gene for cytochrome P-450SU1; the gene for Fd-2 follows the gene for P-450SU2. The deduced amino acid sequences of the two ferredoxins show that, if monomeric, each has a molecular mass of approximately 7 kDa, and alignment of the two sequences demonstrates that they are approximately 52% positionally identical. The spectroscopic properties and iron and acid-labile sulfide contents of both ferredoxins suggest that, as isolated, each contains a single [3Fe-4S] cluster. The presence of only three cysteines in Fd-1 and comparisons with three [4Fe-4S] ferredoxins with high sequence similarity suggest that both Fd-1 and Fd-2 have an alanine in the position where these [4Fe-4S] proteins have a fourth cysteine ligand to the cluster. Transformation of Streptomyces lividans, a strain unable to metabolize sulfonylureas, with DNA encoding both P-450SU1 and Fd-1 results in cells capable of herbicide metabolism. S. lividans transformants encoding only cytochrome P-450SU1 do not metabolize herbicide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of phospholipid and detergent on the substrate specificity of adrenal cytochrome P-450scc. Substrate binding and kinetics of cholesterol side chain oxidation

Cytochrome P-450scc was isolated from mitochondria of bovine adrenal cortex by hydrophobic chromatography on octyl Sepharose followed by affinity chromatography on cholesterol-7-(thiomethyl)carboxy-3 beta-acetate-Sepharose. The partially purified eluate from the octyl Sepharose resin was free of adrenodoxin and adrenodoxin reductase and displayed biphasic binding characteristics for cholesterol, cholesterol sulfate, and cholesterol acetate (CA). Chromatography of the octyl Sepharose eluate on CA-Sepharose removed extraneous proteins and resolved the cytochrome P-450scc into two fractions, each of which displayed monophasic binding with all three substrates. These fractions behaved identically with respect to their ability to bind substrates, their kinetic properties, and their rate of migration during sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The dissociation constants of the cytochrome P-450scc.substrate complexes are 1.1, 2.6, and 1.3 microM for cholesterol, cholesterol sulfate, and cholesterol acetate, respectively. Addition of phospholipids isolated from adrenal cortex mitochondria or adrenodoxin had no effect on the equilibrium binding constants. Addition of Emulgen 913, however, decreased the binding affinities 10-20-fold. Emulgen 913 also inhibited the interaction of adrenodoxin with the cytochrome. An active side chain cleavage system was reconstituted with purified P-450 by addition of saturating amounts of adrenodoxin, adrenodoxin reductase, and NADPH-generating system. The apparent Km values for this reconstituted system of cholesterol, cholesterol sulfate, and cholesterol acetate are 1.8, 1.9, and 0.6 microM, respectively. Since the Km values of substrate oxidation are similar to the Kd values of the cytochrome P-450.substrate complexes, it seems likely that the binding of substrates, particularly when the side chain cleavage system is free of mitochondrial membranes, is not rate-limiting. Based on these results and electrophoretic data, it appears that one cytochrome P-450 present in adrenal mitochondria can oxidize cholesterol, its sulfate, and its acetate. This enzyme represented about 60% of the cytochrome P-450 present in the octyl Sepharose eluate. The factors responsible for the biphasic kinetics of oxidation by intact mitochondria and biphasic binding of sterol substrates by partially purified preparations of cytochrome P-450scc are still unknown.     

Chromosomal assignments of genes coding for components of the mixed-function oxidase system in mice. Genetic localization of the cytochrome P-450PCN and P-450PB gene families and the nadph-cytochrome P-450 oxidoreductase and epoxide hydratase genes

Filter-hybridization studies show that major phenobarbital and pregnenolone-16alpha-carbonitrile-inducible cytochrome P-450 mRNAs in rats were encoded by members of separate, distinct gene families. These gene families are genetically divergent from each and show no cross-hybridization, even under low-stringency conditions. Furthermore, sequences contained in the P-450PB and P-450PCN gene families map to separate chromosomes of the mouse genome. Using mouse X Chinese hamster somatic cell hybrids (EBS cell lines), all distinguishable P-450PCN sequences were found to map to chromosome 6, whereas all P-450PB sequences were located on chromosome 7. Our data support the proposition that the region of the Coh locus on chromosome 7 is the site of the cytochrome P-450PB gene family. The presence of gene families for the cytochromes P-450 occurs in many mammalian species and is likely an important part of the mechanism by which the mixed-function oxidase system is capable of recognizing and metabolizing such a wide array of endogenous and foreign compounds. Conversely, NADPH-cytochrome P-450 oxidoreductase appears to be encoded in many vertebrate species by a single gene and is located on chromosome 6 of the mouse. Corroboratory data are presented to show that the Eph-1 locus on chromosome 1 is the site of at least one microsomal epoxide hydratase gene.     

Characterization and regulation of sex-specific mouse steroid hydroxylase genes

We characterized the genes of the male-specific mouse steroid 16 alpha-hydroxylase (C-P-45016 alpha) and the female-specific mouse steroid 15 alpha-hydroxylase (P-45015 alpha) within two distinct gene families. In spite of the high structural identities within each family, the expression of the hydroxylase genes is uniquely regulated. Moreover, the other family members encode the P-450s which are structurally very similar to the hydroxylases but are not able to catalyze steroid hydroxylase activities. For example, only a single amino acid substitution creates steroid 15 alpha-hydroxylase activity in another family-member P-450coh, which catalyzes coumarin 7-hydroxylase but little steroid hydroxylase activity. It appears, therefore, that the mouse P-450 gene families evolved through gene duplication and selective mutation to create new P-450s structurally as well as to establish novel regulatory elements for the gene expressions.