Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

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.     

Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b5, 200 micrograms/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 micrograms/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6 beta-hydroxytestosterone (51%), 2 beta-hydroxytestosterone (18%), 15 beta-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1 beta-hydroxytestosterone (3%), 16 beta-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16 beta-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited greater than 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin- or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.     

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.     

Monoclonal antibodies distinguish among isozymes of the cytochrome P-450b subfamily

Polyclonal antibody has been shown previously to react identically with cytochromes P-450b and P-450e purified from Long Evans rats and a strain variant of cytochrome P-450b purified from Holtzman rats (P-450bH). In the present study, an array of 12 different monoclonal antibodies produced against cytochrome P-450b has been used to distinguish among these closely related phenobarbital-inducible rat hepatic cytochromes P-450. In immunoblots and enzyme-linked immunosorbent assays, 10 monoclonal antibodies bind to cytochromes P-450b, P-450e, and P-450bH; one monoclonal antibody (B50) recognizes cytochromes P-450b and P-450bH but not cytochrome P-450e; and one monoclonal antibody (B51) is specific for cytochrome P-450b. In addition, one monoclonal antibody (BEF29) reacts strongly with cytochrome P-450f, and another antibody (BEA33) reacts weakly with cytochrome P-450a. No cross-reactions with cytochromes P-450c, P-450d, and P-450g-P-450j were detected with any of the monoclonal antibodies in these assays. Six spatially distinct epitopes on cytochrome P-450b were identified, and differences in antibody reactivity provided evidence for three additional overlapping epitopes. Several monoclonal antibodies are potent inhibitors of testosterone and benzphetamine metabolism supported by cytochrome P-450b in a reconstituted system. B50 and BE52 do not inhibit metabolism of the two substrates by microsomes from untreated rats, but inhibit benzphetamine N-demethylation and testosterone metabolism to 16 alpha- and 16 beta-hydroxytestosterone as well as androstenedione formation 67-94% by microsomes from phenobarbital-treated rats. No other pathways of testosterone metabolism are inhibited by these monoclonal antibodies. The differential inhibition of microsomal metabolism of benzphetamine and testosterone by these monoclonal antibodies is a reflection of the content and inducibility of cytochromes P-450b and P-450e as well as other cytochrome P-450 isozymes.     

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.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Isolation, partial purification, and characterization of the cytochrome P-450-dependent monooxygenase system from the midgut of the earthworm Lumbricus terrestris.

1. Cytochrome P-450 was purified from microsomes of the midgut of the earthworm Lumbricus terrestris up to a maximal specific content of 5.5 nmol P-450/mg protein. 2. At least 3 different cytochromes P-450 with apparent molecular weights of 48,000, 51,000 and 53,000 were identified by SDS-PAGE. 3. Western blot analysis with various polyclonal antibodies did not show structural epitopes common to the cytochromes P-450 of rodents or yeast and L. terrestris. 4. The microsomes contained about 43 pmol P-450/mg protein corresponding to 0.51 nmol P-450/g midgut and 64 pmol P-450/g body weight, respectively, and converted benzyloxyresorufin into resorufin with a Vmax of 2.12 pmol resorufin/min.mg protein and a Km of 770 nM benzyloxyresorufin at 25 degrees C, pH 8.0. 5. The microsomes exhibited a NADPH-cytochrome P-450 reductase activity of 9.4 nmol cytochrome c/min.mg protein. 6. The apparent molecular weight of the threefold-purified reductase was 63,000.     

Comparative characteristics of isolated forms of cytochrome P-450 induced by phenobarbital and perfluorodecalin

Two forms of cytochrome P-450 were isolated from liver microsomes of perfluorodecalin-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from phenobarbital-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromatographic behaviour on 1.8-diaminooctyl-Sepharose 4B and DEAE-Sephacel columns, molecular mass determined by SDS polyacrylamide gel electrophoresis, spectral properties, immunoreactivity, peptide mapping, catalytic activity. These findings suggest that in rat liver microsomes perfluorodecalin and phenobarbital which differ in their chemical structure induce identical forms of cytochrome P-450.     

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid

Cytochrome P-450j has been purified to electrophoretic homogeneity from isoniazid-treated adult male rats; and this enzyme appears to be a major protein induced in hepatic microsomes after administration of isoniazid, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The hemoprotein has a minimum molecular weight of approximately 51,500, and the ferrous-carbonyl complex of cytochrome P-450j has a Soret maximum at 451-452 nm. The oxidized heme iron appears to be predominately in the high spin state as deduced from the Soret maximum at 395 nm. Ethylisocyanide binds to ferrous cytochrome P-450j to yield spectral maxima at approximately 458 and 430 nm with a resultant 458/430 ratio of 0.7 at pH 7.4. Cytochrome P-450j has no measurable catalytic activity for the metabolism of benzo[a]pyrene (3- and 9-hydroxylation), hexobarbital, testosterone, and 5 alpha-androstane-3 alpha,17 beta-diol-3,17-disulfate. Low, but detectable, catalytic activity is obtained for the metabolism of 7-ethoxycoumarin, benzphetamine, p-nitroanisole, zoxazolamine, and 2-hydroxylation of 17 beta-estradiol. In contrast, cytochrome P-450j effectively catalyzes p-hydroxylation of aniline with a turnover of 12.7 nmol/min/nmol cytochrome P-450j. Hydroxyl radical scavengers, Fe-EDTA, superoxide dismutase, and catalase have no effect on aniline p-hydroxylation catalyzed by cytochrome P-450j. Cytochrome P-450j is distinct from nine other rat hepatic microsomal cytochromes P-450 (P-450a-P-450i) previously purified in this laboratory, as well as different isozymes described by other investigators, based on several parameters including minimum molecular weight, spectral properties, and catalytic activity. In Ouchterlony double diffusion plates, antibodies against cytochromes P-450a-P-450f show no cross-reaction with cytochrome P-450j. Structural differences among cytochromes P-450a-P-450j are apparent from the NH2-terminal sequence of cytochrome P-450j, as well as the electrophoretic profiles of proteolytic digests of the hemoproteins.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b^* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b^* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b^* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b^* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase^*. The activity of cytochrome P-450b and P-450b^* increased up to 50% when reconstituted with reductase^* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911.     

Purification and characterization of two constitutive cytochromes P-450 (F-1 and F-2) from adult female rats: identification of P-450F-1 as the phenobarbital-inducible cytochrome P-450 in male rat liver

Two hepatic microsomal cytochromes P-450, P-450F-1 and P-450F-2 were purified to electrophoretic homogeneity from untreated adult female rats by high-performance liquid chromatography (HPLC) with anion-exchange, cation-exchange, and hydroxyapatite columns. Cytochromes P-450F-1 and P-450F-2 were not adsorbed with the anion-exchange column, but were retained on a cation-exchange column and were separated poorly. These forms separated on hydroxyapatite HPLC. The molecular weights of cytochromes P-450F-1 and P-450F-2 were 50,000 and 49,000, respectively. The absolute spectrum of the oxidized forms indicated that they had the low-spin state of heme, and the CO-reduced spectral maxima of cytochromes P-450F-1 and P-450F-2 were at 450 and 448 nm, respectively. Both forms catalyzed the N-demethylation of benzphetamine and had low catalytic activity for 7-ethoxycoumarin. Cytochrome P-450F-1 had low 2 alpha-hydroxylation activity toward testosterone. Cytochrome P-450F-2 had low 15 alpha-hydroxylation activity. On the basis of these results and those of NH2-terminal sequence analysis, cytochrome P-450F-2 seemed to be the typical female-specific cytochrome P-450. The NH2-terminal sequence of cytochrome P-450F-1 was identical to that of cytochrome P-450PB-2 purified from hepatic microsomes of male rats treated with phenobarbital. Cytochromes P-450F-1 and P-450PB-2 had identical chromatographic properties, minimum molecular weight, spectral properties, and peptide maps. Furthermore, the antibody to phenobarbital-inducible cytochrome P-450PB-2 gave a single immunoprecipitin band with cytochrome P-450F-1 by Ouchterlony double-diffusion analysis.     

Comparison of cytochrome P-450 forms induced by phenobarbital and 1,4-bis[3,5-dichloropyridyloxy)]benzene in the mouse and rat liver

A form of cytochrome P-450 (P-450PB) with a molecular weight of 53.5-54.0 kD possessing a high benzphetamine-N-demethylase activity (100-120 nmol formaldehyde/min/nmol cytochrome) was isolated from liver microsomes of phenobarbital-induced C57Bl/6 mice. This cytochrome P-450 form is immunologically identical to its rat liver counterpart-P-450b (Mr = 52 kD) which is also characterized by a high rate of benzphetamine-N-demethylation. It was shown that 1.4-bis[2-(3.5-dichloropyridyloxy])benzene (TCPOBOP) induces in mouse liver the synthesis of the monoxygenase form whose substrate specificity and immunologic properties are identical to those of cytochromes P-450PB and P-450b. The immunochemically quantitated content of this form makes up to 20% of the total P-450 pool in liver microsomes of phenobarbital- or TCPOBOP-induced mice. Immunochemical analysis of microsomes with the use of antibodies to cytochromes P-450PB and P-450b revealed the presence on the electrophoregrams of phenobarbital-induced rat liver microsomes of two immunologically identical forms of cytochrome P-450, i.e., P-450b and P-450e (the latter had a low ability to benzphetamine N-demethylation). Liver microsomes of phenobarbital- or TCPOBP-induced mice gave only one precipitation band corresponding to cytochrome P-450PB.     

Metabolic conditions determining the composition and catalytic activity of cytochrome P-450 monooxygenases in Candida tropicalis.

In the microsomal fraction of Candida tropicalis cells, two distinct monooxygenases were detected, depending on the growth conditions. The distinction of the two monooxygenases was evident from: (i) the absorption maxima in the reduced CO difference spectra of the terminal oxidases (cytochromes P-450 and P-448); (ii) the contents of the monooxygenase components (cytochromes P-450/P-448, NADPH-cytochrome c (P-450) reductase, and cytochrome b5) and (iii) the catalytic activity of the complete system (aliphatic hydroxylation and N-demethylation activity). The occurrence of the respective monooxygenases could be related to the carbon source (n-alkanes or glucose). Oxygen limitation led to a significant increase of cytochrome P-450/P-448 content, independent of the carbon source utilized by the cells. An improved method for the isolation of microsomes enabled us to demonstrate the presence of cytochrome P-448 in glucose-grown cells.     

Purification of hepatic microsomal cytochromes P-450 from beta-naphthoflavone-treated Atlantic cod (Gadus morhua), a marine teleost fish

Four isozymes of cytochrome P-450 were purified to varying degrees of homogeneity from liver microsomes of cod, a marine teleost fish. The cod were treated with beta-naphthoflavone by intraperitoneal injection, and liver microsomes were prepared by calcium aggregation. After solubilization of cytochromes P-450 with the zwitterionic detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propansulfonate, chromatography on Phenyl-Sepharose CL-4B, and subsequently on DEAE-Sepharose, resulted in two cytochrome P-450 fractions. These were further resolved on hydroxyapatite into a total of four fractions containing different isozymes of cytochromes P-450. One fraction, designated cod cytochrome P-450c, was electrophoretically homogeneous, was recovered in the highest yield and constituted the major form of the isozymes. The relative molecular mass of this form (58 000) corresponds well with a protein band appearing in cod liver microsomes after treatment with beta-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56000) showed activity towards 7-ethoxyresorufin in a reconstituted system containing rat liver NADPH-cytochrome P-450 reductase and phospholipid. Differences between these two forms were observed in the rate and optimal pH for conversion of this substrate, and in optical properties. Rabbit antiserum to cod cytochrome P-450c did not show any cross-reactions with cod cytochrome P-450a (Mr 55000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identity with cod cytochrome P-450b (Mr 54000), possibly as a result of contaminating cytochrome P-450c in this fraction.     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

Cytochromes c and P-450 as terminal acceptors in a reconstituted system of mitochondrial hydroxylation

Both cytochromes c and P-450 can be reduced in vitro by a mitochondrial hydroxylation chain consisting of flavoprotein, adrenodoxin and NADPH as electron donors. The effects of pH, ionic strength, content and stoichiometry of intermediate electron carriers on the reduction rate of cytochromes c and P-450 have been compared. The data obtained demonstrate that the reduction of these cytochromes proceeds by different ways. It has been found that cytochrome c has an inhibitory effect on cytochrome P-450 reduction. On the other hand, cytochrome P-450 remarkably activates the process of cytochrome c reduction.     

Isolation and characterization of the basic forms of cytochrome P-450 from liver microsomes of C57BL mice after phenobarbital and 3-methylcholanthrene induction

Using hydrophobic and ion-exchange chromatography, cytochromes P-450 and P-448 from liver microsomes of C 57 BL mice induced by phenobarbital and 3-methylcholantrene were isolated. The cytochromes purified to homogeneity as evidenced from SDS polyacrylamide gel electrophoresis were characterized in terms of molecular weight and catalytic and spectral properties and by peptide mapping. Cytochrome P-450, in contrast to cytochrome P-448, was not bound to the ion-exchanger and was eluted in a void volume. Cytochrome P-450 (Mr = 51 000) elicits a low spin signal and reveals a high catalytic activity toward aminopyrine and a low catalytic activity toward benz(a)pyrene. Cytochrome P-448 (Mr = 55 000) elicits both high an low spin signals and reveals a high catalytic activity toward benz(a)pyrene and a low catalytic activity toward aminopyrine. Limited proteolysis with papain demonstrated the differences in the proteins primary structure.     

Induction of cytochrome P-450 forms by prolonged administration of nifedipine

In experiments on male Wistar rats it has been found that nifedipine applied in a dose of 10 mg/kg body weight i.p. daily for 20 days did not significantly increase the total amount of cytochrome P-450 but markedly increased the 7 alpha-, 16 beta- and 6 beta-hydroxylation of androstenedione in liver microsomes, suggesting the induction of cytochromes P-450a, P-450b and P-450p respectively. The induction of cytochrome P-450b was also confirmed immunochemically with polyclonal antibodies against cytochrome P-450b/e.     

Characterization of rat and human liver microsomal cytochrome P-450 forms involved in nifedipine oxidation, a prototype for genetic polymorphism in oxidative drug metabolism

The metabolism of the dihydropyridine calcium antagonist and vasodilator nifedipine has been reported to exhibit polymorphism among individual humans (Kleinbloesem, C. H., van Brummelen, P., Faber, H., Danhof, M., Vermeulen, N. P. E., and Breimer, D.D. (1984) Biochem. Pharmacol. 33, 3721-3724). Nifedipine oxidation has been shown to be catalyzed by cytochrome P-450 (P-450) enzymes. Reconstitution, immunoinhibition, and induction studies with rat liver indicated that the forms designated P-450UT-A and P-450PCN-E are the major contributors to microsomal nifedipine oxidation. The P-450 which oxidizes nifedipine (P-450NF) was purified to electrophoretic homogeneity from several human liver samples. Antibodies raised to P-450NF were highly specific as judged by immunoblotting analysis and inhibited greater than 90% of the nifedipine oxidase activity in human liver microsomes. A monoclonal antibody raised to the human P-450 preparation reacted with both human P-450NF and rat P-450PCN-E. Immunoblotting analysis of 39 human liver microsomal samples using anti-P-450NF antibodies revealed the same 52,000-dalton polypeptide, corresponding to P-450NF, with only one of the microsomal samples showing an additional immunoreactive protein. The level of nifedipine oxidase activity was highly correlated with the amount of P-450NF thus detected using either polyclonal (r = 0.78) or monoclonal (r = 0.65) antibodies, suggesting that the amount of the P-450NF polypeptide may be a major factor in influencing the level of catalytic activity in humans as well as rats. Cytochrome b5 enhanced the catalytic activity of reconstituted P-450NF, and anti-cytochrome b5 inhibited nifedipine oxidase activity in human liver microsomes. P-450NF also appears to be a major contributor to human liver microsomal aldrin epoxidation, d-benzphetamine N-demethylation, 17 beta-estradiol 2- and 4-hydroxylation, and testosterone 6 beta-hydroxylation, the major pathway for oxidation of this androgen in human liver microsomes.