Purification of human liver cytochrome P-450 and comparison to the enzyme isolated from rat liver

Human liver cytochrome P-450 was isolated from autopsy samples using cholate extraction and chromatography on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE-cellulose gels. Purified preparations contained as much as 14 nmol cytochrome P-450 mg^?1 protein, were free of other hemoproteins, and were active in the mixed-function oxidation of d-benzphetamine and 7-ethoxycoumarin when coupled with either rat or human liver NADPH-cytochrome P-450 reductase. Some of the preparations were apparently homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; apparent subunit M_rs estimated for several preparations were 53,000 or 55,500. The amino acid composition of one preparation was determined and found to resemble those of rat liver cytochromes P-450, although some variations were noted. Rabbit antibodies raised to phenobarbital-treated rat liver cytochrome P-450 were more effective in inhibiting d-benzphetamine N-demethylase activity in human liver microsomes than were antibodies raised to 3-methylcholanthrene-treated rat liver cytochrome P-450. These antibodies also inhibited benzo(a)pyrene hydroxylation in human liver microsomes, although the inhibition patterns did not follow a general pattern as in the case of benzphetamine demethylase activity. Microsomes prepared from three different human liver samples were more effective in eliciting complement fixation with antibodies raised to phenobarbitalthan to 3-methylcholanthrene-treated rat liver cytochrome P-450. Complement fixation in such systems appears to result from similarity of certain rat and human liver cytochrome P-450 antigenic determinants, as fixation could be inhibited by removal of cytochrome P-450-directed antibodies from the total immunoglobulin population and purified human cytochrome P-450 was more effective (on a protein basis) than liver microsomes in producing fixation. Human liver microsomes prepared from five different individuals all produced ≥ 90% complement fixation, but variations were observed in the fixation curves plotted either versus microsomal protein or versus spectrally detectable microsomal cytochrome P-450.These results indicate that human liver microsomal cytochromes P-450 can be isolated using modifications of techniques developed for laboratory animals and that human and rat liver cytochromes P-450 share certain features of structural, functional, and immunological similarity. The available data suggest the existence of multiple forms of human liver microsomal cytochrome P-450, but possible artifacts associated with the use of autopsy samples suggest caution in advancing such a conclusion.     

Purification of characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls

A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450_e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450_b and P-450_c, have also been highly purified during the isolation of cytochrome P-450_e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450_b, highly purified cytochrome P-450_e is immunochemically identical to cytochrome P-450_b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 (P-450_a, P-450_c, P-450_d) or epoxide hydrolase. Purified cytochrome P-450_e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450_b or P-450_d (52,000) but clearly distinct from cytochromes P-450_a (48,000) and P-450_c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450_e is at 450.6 nm, whereas the peak of cytochrome P-450_b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450_e and P-450_b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450_b and P-450_e, respectively. Metyrapone binds to reduced cytochromes P-450_e and P-450_b (absorption maximum at 445–446 nm) but not cytochromes P-450_a, P-450_c, or P-450_d. Metabolism of several substrates catalyzed by cytochrome P-450_e or P-450_b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450_e usually paralleled that of cytochrome P-450_b except that the rate of metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450_e was only 15–25% that of cytochrome P-450_b. In contrast, cytochrome P-450_e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450_b. Cytochrome P-450_d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450_a, P-450_b, P-450_c, or P-450_e. The peptide fragments of cytochromes P-450_e and P-450_b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.     

Covalent binding of benzo[a]pyrene to cytochrome P-450βNF-B2 and other proteins in reconstituted mixed-function oxidase systems

A reconstituted mixed-function oxidase system, containing the major β-naphthoflavone-induced isozyme of rat liver cytochrome P-450 bound benzo[a]pyrene covalently in the presence of NADPH. NADPH-cytochrome P-450 reductase was required for binding and a maximum rate of adduct formation was obtained at 8 units of reductase per nmol cytochrome P-450. Phosphatidylcholine inhibited this reaction. Benzo[a]pyrene was bound to the cytochrome, but not to the reductase, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Approximately 6 molecules of benzo[a]pyrene bound to each molecule cytochrome P-450 during prolonged incubations. No binding occurred when the β-naphthoflavone-induced isozyme of cytochrome P-450 was replaced by the major isozyme induced by phenobarbital, but both cytochromes incorporated benzo[a]pyrene to approximately the same extent when they were incubated together in the presence of the reductase and NADPH. Metabolically activated benzo[a]pyrene also bound covalently to purified epoxide hydrodrolase, when this enzyme was added to the reconstituted mixed-function oxidase system.     

The effects of carbon disulphide on rat liver microsomal mixed-function oxidases, in vivo and in vitro

An intraperitoneal dose of CS₂ (500mg/kg) to male rats resulted in loss of liver microsomal mixed-function-oxidase activity (85% loss of biphenyl 4-hydroxylase), followed by denaturation of liver cytochrome P-450 to cytochrome P-420, and degradative loss of both cytochromes (50% loss). Losses of NADPH–cytochrome c reductase (20%) and cytochrome b₅ were considerably less. Intraperitoneal administration of CS₂ (100mg/kg) to rats pretreated wtih phenobarbitone or 3-methylcholanthrene resulted in similar losses, but the rate of destruction was greater with cytochrome P-450 than with cytochrome P-448. At 12h after intraperitoneal injection of CS₂ to non-pretreated rats, a new cytochrome (P-448) appeared. Rat liver microsomal preparations incubated with CS₂ in the presence of NADPH and O₂ resulted in loss of cytochrome P-450 and mixed-function-oxidase activity directly related to the concentration of CS₂ (10–100μm) and to the period of incubation. Addition of EDTA (1mm) completely inhibited this destruction of cytochrome P-450 by CS₂ in vitro. Addition of CS₂ to liver microsomal preparations resulted in moderate increases in the K_s values for type-I or type-II substrates, but these were insufficient to account for the inhibition of the mixed-function oxidases. We therefore suggest that desulphuration of CS₂ leads to binding of the S to cytochrome P-450, denaturation of cytochrome P-450 to cytochrome P-420, and ultimately to destruction of these cytochromes by autoxidation.     

Comparative study of two highly purified forms of liver microsomal cytochrome P-450: circular dichroism and other properties.

The two major forms of rabbit liver microsomal cytochrome P-450, P-450_LM2 and P-450_LM4, which were previously shown to differ in their absorption spectra, electrophoretic and immunochemical properties, and substrate specificities, have been further characterized by several methods, (a) The two cytochromes have different CD spectra in the ferric state but similar spectra when reduced. Upon conversion of P-450_LM2 to P-420 by treatment with sodium dodecyl sulfate, the CD spectrum is greatly diminished except in the far ultraviolet region, whereas the conversion of P-450_LM4 toP-420 with this detergent results in a spectrum with a new positive band in the visible region, (b) Although P-450_LM4 has a much higher tryptophan content than P-450_LM2, the fluorescence spectra of these proteins are similar in magnitude. Upon denaturation, the fluorescence of P-450_LM4 increases, thereby indicating a large quenching effect in the native protein, (c) Studies on the interaction of dilauroylglyceryl-3-phosphorylcholine with the cytochromes showed that P-450_LM2 gives a much stronger Type I difference spectrum than does P-450_LM4. This phospholipid has no significant effect on the state of aggregation of these cytochromes as judged by calibrated gel filtration. The CD spectra of P-450_LM2 and P-450_LM4 are unchanged in the visible region but are enhanced in the far ultraviolet region upon the addition of phosphatidylcholine. The results appear to indicate an increase in α-helical content, particularly with P-450_LM4, in the presence of the phospholipid.     

Covalent binding of polychlorinated biphenyls to proteins by reconstituted monooxygenase system containing cytochrome P-450

Incubation in the presence of NADPH and molecular oxygen of ¹⁴C-labeled polychlorinated biphenyls (PCBs) and two tetrachlorobiphenyl (TCB) isomers with a reconstituted system containing NADPH-cytochrome P-450 reductase and cytochrome P-450, both purified from liver microsomes of phenobarbital(PB)-pretreated rabbits, led to covalent binding of radioactive metabolites of PCBs and TCBs to the protein components of the system. A rabbit liver cytosol fraction added to the system provided more binding sites for the activated metabolites and thus increased the extent of binding markedly. The binding reaction depended absolutely on the reductase, cytochrome P-450 and NADPH, and required dilauroyl phosphatidylcholine and sodium cholate for maximal activity. A further stimulation of the binding was attained by including cytochrome b₅ in the reconstituted system. Four forms of cytochrome P-450, purified from liver microsomes of PB- and 3-methylcholanthrene(MC)-treated rabbits and rats, were used to reconstitute the PCB- and TCB-metabolizing systems, and it was found that PB-inducible forms of the cytochrome from both animals were more active than those inducible by MC in catalyzing the PCB- and TCB-binding reaction. Sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis indicated that, in the system containing the reductase, cytochrome P-450 and cytochrome b₅, PCB metabolites bound to the reductase and cytochrome P-450, but not to cytochrome b₅. In the presence of the liver cytosol fraction, the binding took place to many cytosolic proteins in addition to the reductase and cytochrome P-450.     

Purification and immunochemical characterization of the two adrenal cortex mitochondrial cytochrome P-450-proteins.

A procedure for the separation and purification of two distinct P-450 cytochromes, termed P-450_scc and P-450_11β, solubilized from bovine adrenal cortex mitochondria is reported. Important features of the purification procedure are uses of aniline-substituted Sepharose chromatography and utilization of the markedly different characteristics in stability and solubility of each cytochrome. Polyacrylamide gel electrophoresis of the two purified preparations in sodium dodecyl sulfate reveals single protein bands. The P-450_scc, which catalyzes the formation of pregnenolone from cholesterol, has a turnover number of 16 mol of pregnenolone formed per minute per mole of P-450-heme. The P-450_11β catalyzes the hydroxylation of deoxycorticosterone at 11β- and 18-positions with turnover numbers of 110 and 18, and of 4-androstene-3,17-dione at 11β- and 19-positions with turnover numbers of 41 and 12, respectively. The recoveries of the P-450_scc and P-450_11β, in terms of the catalytic activities, are 20% and 15%, respectively, from the crude extract which contains the two activities in a ratio of roughly 2:1. Each rabbit antibody prepared against the two purified P-450-proteins interacts with respective, but not alternative cytochrome P-450, whether in the crude mitochondrial preparation or in the purified preparation. The observed patterns of immunoprecipitation and inhibition of catalytic activity indicated that the two P-450 proteins are immunochemically different from each other. Neither antibody immunoprecipitates with a highly purified bacterial cytochrome P-450, P-450_cam.     

In vitro biosynthesis of liver cytochrome P450 mature peptide sub-unit by translation of isolated poly(A)+ mRNA from normal and phenobarbital induced rats

The biosynthesis of a cytochrome P₄₅₀ peptide sub-unit by the in vitro translation of total hepatic poly (A)⁺ mRNA in an heterologous cell-free-system is described. The ability of the liver poly (A)⁺ RNA preparations from normal and phenobarbital induced rats to promote protein synthesis and the identification of in vitro synthesized proteins revealed the presence of a cytochrome P₄₅₀ peptide sub-unit presenting the same apparent molecular weight of the native peptide. This fact demonstrates that rat liver poly (A)⁺ mRNA fraction contains an important amount of cytochrome P₄₅₀ peptide messages. Total poly (A)⁺ RNA from rats in an early phenobarbital induction stage exhibits a higher cytochrome P₄₅₀ template activity in good agreement with the enhancement of this hemeprotein concomitantly observed in vivo, in the liver microsomes, it is also concluded that cytochrome P₄₅₀, peptide sub-unit, induced in rat liver by phenobarbital, is translated in its mature form.     

Isolation and characterization of a constitutive form of rabbit liver microsomal cytochrome P-450

A heretofore unrecognized form of cytochrome P-450 was purified from rabbit liver microsomes with an average yield and purity similar to that of other highly purified forms of cytochrome P-450. Several properties of this cytochrome are contrasted with those of form 2, the major phenobarbital-inducible cytochrome P-450, form 4, the major 2,3,7,8-tetrachlorodibenzo-p-dioxin-inducible cytochrome, and form 6, a cytochrome that is selectively induced in liver microsomes by 2,3,7,8-tetrachlorodibenzo-p-dioxin during the perinatal period. Thes four forms can be distinguished by virtue of their molecular weights as determined using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, by their respective peptide fingerprints, and by the monospecificity of their antisera. Since the enumerated properties are thought to reflect the primary structure of the cytochromes and since the observed differences are extensive, we suggest that these four forms are not derived from a common protein precursor.     

NADPH-cytochrome P-450 reductase of yeast microsomes.

NADPH-cytochrome c reductase of yeast microsomes was purified to apparent homogeneity by solubilization with sodium cholate, ammonium sulfate fractionation, and chromatography with hydroxylapatite and diethylaminoethyl cellulose. The purified preparation exhibited an apparent molecular weight of 83,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reductase contained one molecule each of flavin-adenine dinucleotide and riboflavin 5′-phosphate, though these were dissociative from the apoenzyme. The purified reductase showed a specific activity of 120 to 140 μmol/min/mg of protein for cytochrome c as the electron acceptor. The reductase could reduce yeast cytochrome P-450, though with a relatively slow rate. The reductase also reacted with rabbit liver cytochrome P-450 and supported the cytochrome P-450-dependent benzphetamine N-demethylation. It can, therefore, be concluded that the NADPH-cytochrome c reductase is assigned for the cytochrome P-450 reductase of yeast. The enzyme could also reduce the detergent-solubilized cytochrome b₅ of yeast. So, this reductase must contribute to the electron transfer from NADPH to cytochrome b₅ that observed in the yeast microsomes.     

Immunological evidence for phenobarbital-stimulated synthesis of cytochrome P-450 in primary cultures of chick embryo hepatocytes

The induction of cytochrome P-450 by phenobarbital was studied in primary cultures of chick embryo hepatocytes. The rate of the de novo synthesis of the induced form of cytochrome P-450 was measured directly and specificially, using form-specific anti-cytochrome antibodies that quantitatively immunoprecipitated this form from the radiolabeled hepatocytes. Additionally, the steady-state levels of the cytochrome were estimated spectrophotometrically and electrophoretically. In the presence of phenobarbital the synthesis of cytochrome P-450_PB by cultured hepatocytes was markedly accelerated. Furthermore, the same cytochrome P-450_PB form was induced by phenobarbital in vivo in chicken liver and in the cultured chick embryo hepatocytes. Their identity was judged from immunological and electrophoretic properties of these induced cytochromes. Immunological cross-reactivity was also detected between the cytochrome P-450_PB forms from chick embryo hepatocytes and from adult rat liver. The immunological cross-reactivity observed between the phenobarbital-induced cytochrome P-450 forms from different species was not observed between the different cytochrome forms with the same liver (Thomas, P.E., Reik, L.M., Ryan, D.E. and Levin, W. (1981) J. Biol. Chem. 256, 1044–1052). Implications as to the evolutionary origin of the different cytochrome forms are discussed.     

Cytochrome P-450 from mitochondria of bovine adrenal cortex Comparison of cholesterol side-chain cleavage P-450 with steroid 11β-hydroxylation P-450 and immunochemical cross-reactivity between adrenal mitochondrial and liver microsomal cytochromes P-450

Adrenocortical mitochondrial cytochrome P?450 specific to the cholesterol side-chain cleavage (desmolase) reaction differs from that for the 11β-hydroxylation reaction of deoxycorticosterone. The former cytochrome appears to be more loosely bound to the inner membrane than the latter. Upon ageing at 0°C or by aerobic treatment with ferrous ions, the desmolase P-450 was more stable than the 11β-hydroxylase P-450. By utilizing artificial hydroxylating agents such as cumene hydroperoxide, H₂O₂, and sodium periodate, the hydroxylation reaction of deoxycorticosterone to corticosterone in the absence of NADPH was observed to a comparable extent with the reaction in the presence of adrenodoxin reductase, adrenodoxin and NADPH. However, the hydroxylation reaction of cholesterol to pregnenolone was not supported by these artificial agents.Immunochemical cross-reactivity of bovine adrenal desmolase P-450 with rabbit liver microsomal P-450_LM4 was also investigated. We found a weak but significant cross-reactivity between the adrenal mitochondrial P-450 and liver microsomal P-450_LM4, indicating to some extent a homology between adrenal and liver cytochromes P-450.     

Properties of electrophoretically homogeneous phenobarbital-inducible and beta-naphthoflavone-inducible forms of liver microsomal cytochrome P-450.

Procedures are described for the isolation of two forms of rabbit liver microsomal liver microsomal cytochrome P-450 (P-450LM) in homogeneous state. They are designated by their relative electrophoretic mobilities on polyacrylamide gel in the presence of sodium dodecyl sulfate as P-450LM2 and P-450LM4. P-450LM2, which was isolated from phenobarbital-induced animals, has a subunit molecular weight of 48,700. The best preparations contain 20 nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. P-450LM4, which is induced by beta-naphthoflavone but is also present in phenobarbital-induced and untreated animals, was isolated from all three sources and found to have a subunit molecular weight of 55,300. The best preparations contain 17nmol of the cytochrome per mg of protein and 1 molecule of heme per polypeptide chain. Some of the purified preparations of the cytochromes, although electrophoretically homogeneous, contain apoenzyme due to heme loss during purification. The purified proteins contain no detectable NADPH-cytochrome P-450 reductase, cytochrome b5, or NADH-cytochrome b5 reductase, and only low levels of phospholipid (about 1 molecule per subunit). Amino acid analysis indicated that P-450LM2 and P-450LM4 are similar in composition, but the latter protein has about 60 additional residues. The COOH-terminal amino acid of P-450LM2 is arginine, as shown by carboxypeptidase treatment, whereas that of P-450LM4 is lysine. NH2-terminal amino acid residues could not be detected. Carbohydrate analysis indicated that both cytochromes contain 1 residue of glucosamine and 2 of mannose per polypeptide subunit. The optical spectra of the oxidized and reduced cytochromes and carbon monoxide complexes were determined. Oxidized P-450LM2 has maxima at 568, 535, and 418 nm characteristic of a low spin hemeprotein, and P450LM4 from beta-naphthoflavone-induced, phenobarbital-induced, or control microsomes has maxima at 645 and 394 nm, characteristic of the high spin state. The spectrum of -450lm4 becomes similar to that of P-450LM2 at high protein concentrations or upon the addition of detergent (Renex), whereas the spectrum of P-450LM2 is unaffected by the protein concentration or the presence of detergent. Electron paramagnetic resonance spectrometry of the purified cytochromes indicated that oxidized -450lm2 is in the low spin state, whereas P-450LM4 is largely, but not entirely, in the high spin state.     

Mechanism of inhibition of microsomal mixed-function oxidation by the gut-contents inhibitor of the southern armyworm (Prodenia eridania)

A potent inhibitor of microsomal mixed-function oxidation reactions in insects had previously been isolated and partially purified from the gut contents of Prodenia eridania and shown to be associated with proteinase activity. Incubation of rat liver microsomal fraction with low concentrations of this inhibitor led to solubilization of NADPH–cytochrome c reductase, which was paralleled by the inactivation of reduction of cytochrome P-450 by NADPH and by the inhibition of NADPH-linked benzo[3,4]pyrene hydroxylation and aminopyrine demethylation. There was little or no effect on cytochromes b₅ and P-450, nor was the capacity of the latter catalyst to combine with exogenous substrates decreased. Contrary to the findings with NADPH, preincubation of microsomal fraction with the inhibitor did not cause a significant decrease in the rate of cytochrome P-450 reduction by NADH, supporting the assumption that different catalysts are involved in the electron transfer from NADH and NADPH to cytochrome P-450. The findings indicate the importance of taking the possible presence of endogenous inhibitors into consideration when evaluating low or absent mixed-function oxidation activities found in insect systems in vitro.     

Purification and characterization of the cytochrome P-448 component of a benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae

Cytochrome P-448, a type of cytochrome P-450, from brewer's yeast (Saccharomyces cerevisiae) grown under conditions of glucose repression was isolated and purified. Triton X-100 in very low concentration proved to be very effective in stabilizing P-448 in the microsomal fraction and later prevented its conversion to cytochrome P-420 through solubilization with various ionic and nonionic detergents. Highest yields were obtained with 1% sodium cholate, in the presence of 0.1% Triton X-100 and reduced glutathione. A novel combination of hydrophobic adsorption and other chromatographic techniques was used for the purification of cytochrome P-448. These involve the use of amino octyl-Sepharose 4B, instead of the low-yielding aminohexyl derivative, followed by the fast-running hydroxyapatite-cellulose column. Finally, the use of DEAE-Sephacel was found to increase greatly the purity of the cytochrome P-448 obtained. The molecular weight of this preparation was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M_r, 55,500). Using the known molar extinction coefficient of the carbon monoxide-difference spectrum the estimate of degree of purity of cytochrome P-448 obtained by this purification procedure was between 88 and 97%. Electrophoresis also showed that this preparation was completely homogeneous and assays showed that it was also completely free of cytochrome b_s, cytochrome c reductase and cytochrome P-420. Purified cytochrome P-448 reconstituted with cytochrome P-450 (cytochrome c) reductase, isolated from yeast, showed 10-fold higher aryl hydrocarbon hydroxylase activity with benzo[a]pyrene as a substrate than the corresponding microsomal fraction enzyme. Kinetics of benzo[a]pyrene hydroxylation were determined: K_m (33 μm) was comparable with that reported for purified hepatic cytochrome P-448. The number of binding sites of microsomal and purified cytochromes P-450 (from liver of phenobarbital-induced rats) and yeast cytochrome P-448 with benzo[a]pyrene has been determined using and equilibrium gel filtration method. There is one binding site in each case (contrast with six sites for microsomal enzymes). The Scatchard plot gives number of binding sites, apparent association constants (K), and the equivalent dissociation constants (K_s). Comparison is made with spectral dissociation constants for these enzymes and benzo[a]pyrene. Thus the proportion bound, dissociation constant (K_s), and stoichiometry of rat liver (phenobarbital induced) and yeast cytochrome P-448 with benzo[a]pyrene were compared with corresponding values for microsomal fractions of both systems. Purified enzymes had higher K_s values in both cases, and the proportion of enzyme that bound benzo[a]pyrene was high (53%) for liver and this value is 100% for purified enzyme from yeast, which is the same as the value obtained for the microsomal enzyme from yeast.     

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.     

A simple and rapid procedure for the purification of phenobarbital-inducible cytochrome P-450 from rat liver microsomes.

A rapid and simple procedure has been developed for the purification of a phenobarbital-inducible form of cytochrome P-450 from the liver microsomes of phenobarbitalpretreated rats. Within 2 days approximately 1000–1500 nmol of highly purified cytochrome P-450 with a specific content of 16 nmol/mg protein can be recovered from 4 g of microsomal protein. The procedure consists of solubilization of microsomal protein with sodium cholate, fractionation with polyethylene glycol, and column chromatography at room temperature on DEAE-cellulose. The resulting DEAE-cellulose fraction electrophoreses on polyacrylamide gels in the presence of sodium dodecyl sulfate as a major protein band with a minimum molecular weight of 52,000 and a few faint bands. Further chromatography on QAE Sephadex A-25 essentially removes these faint bands and increases the specific content slightly to 17 nmol/mg protein. Relatively low amounts of this form of cytochrome P-450 appear to be present in microsomes of untreated rats since less than 1% can be recovered as the DEAE-cellulose fraction by this procedure. An identical form is inducible by phenobarbital in rats of different ages and sex. In a reconstituted system under optimal assay conditions, this form of cytochrome P-450 catalyses the N-demethylation of benzphetamine with a turnover number greater than 100 and hydroxylates testosterone at the 16α position but not at the 6β or 7α position.     

K+-valinomycin and chloride conductance of the human red cell membrane. Influence of the membrane protonophore carbonylcyanide m-chlorophenylhydrazone

The major form of microsomal cytochrome P-450 induced by trans-stilbene oxide in the liver of male Sprague-Dawley rats was purified and characterized, and compared with the isolated cytochrome P-450 B2 forms from phenobarbital- and 3-methylcholanthrene-pretreated animals. The apparent subunit molecular weight of the trans-stilbene oxide-induced cytochrome was found to be 53 000 using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the absorbance maximum of the carbon monoxide complex of the ferrous cytochrome was 450 nm. Reconstitution of the N-demethylase activity towards three different substrates showed high and similar activities with the cytochrome P-450 B2 forms from trans-stilbene oxide or phenobarbital-treated rats, with one exception. Amino-acid analysis also showed a very high degree of similarity between these two forms. Upon proteinase treatment with three different proteinases the trans-stilbene oxide-induced cytochrome demonstrated in each case a peptide pattern identical to that obtained with the phenobarbital-induced B2 form. Furthermore, both forms are completely immunologically cross-reactive. We therefore conclude from these experiments that the liver microsomal P-450 B2 from trans-stilbene oxide and phenobarbital-treated rats are very closely related, if not identical.     

Interaction of epoxide hydrolase with itself and other microsomal proteins

The interactions of rat liver epoxide hydrolase (EC 3.3.2.3) with itself and with cytochromes P-450 and NADPH-cytochrome P-450 reductase were investigated in microsomal preparations and in reconstituted systems in which all of the enzymes are functionally active. Hydrodynamic measurements indicated that purified epoxide hydrolase behaves as a single aggregate of approximately 16 monomeric units and that further aggregation of the protein only occurs in the presence of high concentrations of phospholipid. Neither guanidine-HCl nor the nonionic detergent Lubrol PX was able to completely dissociate the aggregate into monomers. The interactions of epoxide hydrolase with NADPH-cytochrome P-450 reductase and the major forms of cytochrome P-450 isolated from phenobarbital- and 5,6-benzoflavone-treated rats were studied by Soret difference spectroscopy, by perturbation of the fluorescence of NADPH-cytochrome P-450 reductase and fluorescein-labeled epoxide hydrolase, and by CD spectroscopy. The spectra provided evidence that binding of the proteins to each other occurs and some of the results suggest that affinity constants are on the order of 10⁷, m^?1. The spectral perturbations were not observed with other intrinsic membrane proteins. When microsomes were treated with the crosslinking reagent dimethylsuberimidate and solubilized with detergents, epoxide hydrolase could be precipitated with antibodies raised to cytochromes P-450 or NADPH-cytochrome P-450 reductase. Transient times were determined for the conversion of 1-octene to octene-1,2-dihydrodiol in a reconstituted enzyme system and for the conversion of naphthalene to naphthalene-1,2-dihydrodiol in rat liver microscomes and compared to the transient times predicted from the enzymatic rates of hydrolysis of the intermediate epoxides. In all cases the observed transient times were shorter than expected, in support of the view that coupling of epoxide hydrolase with cytochromes P-450 occurs. These results support the view that epoxide hydrolase couples with cytochrome P-450-containing mixed-function oxidase systems and may have relevance to the metabolism of potentially harmful xenobiotics by these enzymes.     

Reconstitution of the reduced nicotinamide adenine dinucleotide phosphate- and reduced nicotinamide adenine dinucleotide-peroxidase activities from solubilized components of rat liver microsomes.

The liver microsomal enzyme system that catalyzes the oxidation of NADPH by organic hydroperoxides has been solubilized and resolved by the use of detergents into fractions containing NADPH-cytochrome c reductase, cytochrome P-450 (or P-448), and microsomal lipid. Partially purified cytochromes P-450 and P-448, free of the reductase and of cytochrome b₅, were prepared from liver microsomes of rats pretreated with phenobarbital (PB) and 3-methylcholanthrene (3-MC), respectively, and reconstituted separately with the reductase and lipid fractions prepared from PB-treated animals to yield enzymically active preparations functional in cumene hydroperoxide-dependent NADPH oxidation. The reductase, cytochrome P-450 (or P-448), and lipid fractions were all required for maximal catalytic activity. Detergent-purified cytochrome b₅ when added to the complete system did not enhance the reaction rate. However, the partially purified cytochrome P-450 (or P-448) preparation was by itself capable of supporting the NADPH-peroxidase reaction but at a lower rate (25% of the maximal velocity) than the complete system. Other heme compounds such as hematin, methemoglobin, metmyoglobin, and ferricytochrome c could also act as comparable catalysts for the peroxidation of NADPH by cumene hydroperoxide and in these reactions, NADH was able to substitute for NADPH. The microsomal NADH-dependent peroxidase activity was also reconstituted from solubilized components of liver microsomes and was found to require NADH-cytochrome b₅ reductase, cytochrome P-450 (or P-448), lipid, and cytochrome b₅ for maximal catalytic activity. These results lend support to our earlier hypothesis that two distinct electron transport pathways operate in NADPH- and NADH-dependent hydroperoxide decomposition in liver microsomes.