Rat Liver NADPH-cytochrome P-450 Reductase Research Articles

Purification and characterization of a soluble form of rat liver NADPH-cytochrome P-450 reductase highly expressed in Escherichia coli

A recombinant cDNA of rat liver NADPH-cytochrome P-450 reductase (CPR), which lacks the N-terminal hydrophobic region, was amplified by PCR and cloned. The N-truncated cDNA named tCPR was ligated into a pBAce vector and expressed. The tCPR protein expressed in Escherichia coli was recovered into the soluble fraction of the cell lysate and purified to homogeneity by three sequential purification procedures; (I) anion-exchange chromatography on a DEAE-cellulose (DE-52) column, (II) affinity chromatography on 2 ′,5 ′-ADP Sepharose 4B, and (III) chromatography on a hydroxyapatite column. The average yield was 47 mg per liter of culture medium. The absorption spectrum of the purified tCPR protein was identical to that of a native full-length CPR purified from rat liver, indicating that tCPR also possesses one molecule each of FAD and FMN. The tCPR protein was able to reduce cytochrome c and was also able to assist heme degradation by a soluble form of rat heme oxygenase-1. However, it failed to support the O-deethylation of 7-ethoxycoumarin by cytochrome P-450 1A1, indicating that the presence of the N-terminal hydrophobic domain is necessary for CPR to interact with cytochrome P-450. Previously, to prepare a soluble form of CPR, full-length CPR was treated with proteinases that selectively removed the N-terminal domain. With the expression system established in this study, however, the soluble and biologically active tCPR protein can be readily prepared in large amounts. This expression system will be useful for mechanistic as well as structural studies of CPR.

Mutagenesis Study of Asp-290 in Cytochrome P450 2B11 Using a Fusion Protein with Rat NADPH-Cytochrome P450 Reductase

Asp-290 of the phenobarbital-inducible dog liver cytochrome P450 (P450) 2B11 was mutated to nine other amino acid residues by site-directed mutagenesis, and the functional significance of the unique negative charge in P450 2B11 at that position was studied. To facilitate the analysis of mutated P450 2B11 enzymes heterologously expressed inEscherichia coli,an enzymatically active fusion enzyme was genetically engineered between the cDNAs for P450 2B11 and rat liver NADPH-cytochrome P450 reductase using a Ser-Thr linker as previously described (Fisheret al.,1992,Proc. Natl. Acad. Sci. USA89, 10817–10821). Sonicated whole-cell lysates ofE. colicells expressing the wild-type fusion protein were able to catalyze the 16-hydroxylation of androstenedione (AD) in the absence of added reductase, and exhibited activities and androstenedione metabolite profiles very similar to those of purified and reconstituted enzyme preparations. The substitution of Ala, Glu, Gly, Met, Asn, Arg, Ser, Thr, or Val for Asp-290 of P450 2B11 resulted in decreased AD hydroxylase activities as assessed using solubilized membranes. Replacement of Asp-290 with Glu yielded the highest activity (55% of wild type), while substituting the positively charged amino acid Arg created an enzyme with the lowest activity (<1% of wild-type activity). Regioselectivity of AD hydroxylation was not affected although the stereoselectivity of hydroxylation at the 16 carbon position was altered in some cases. The use of the fused enzyme to study the effects of site-directed mutagenesis has resulted in the demonstration of the importance of size and charge at position 290 for enzymatic activity of P450 2B11.

Understanding the structural aspects of neuronal nitric oxide synthase (NOS) using microdissection by molecular cloning techniques: molecular dissection of neuronal NOS.

The neuronal isoform (Type I) of nitric oxide synthase (NOS) requires Ca+2/calmodulin to catalyze the formation of NO● and citrulline from L-arginine (1) and molecular oxygen (2) with reducing equivalents from NADPH. The overall reaction is a five-electron process involving two successive monooxygenation steps with the obligatory formation of N-hydroxy-L-arginine as the oxygenated intermediate. The neuronal NOS shares with the other two known isoforms the unusual property, for a mammalian enzyme, of containing iron protoporphyrin IX (3–6), FAD and FMN (3, 7–9), and tetrahydrobiopterin (9). Bredt, et al. (10) had previously demonstrated the remarkable (58%) sequence similarity of rat brain NOS to rat liver NADPH-cytochrome P450 reductase and determined that the C-terminal 641 amino acids of NOS display consensus regions for both flavin and nucleotide binding. It was reported that carbon monoxide (CO) inhibited both macrophage and neuronal NOS activity (3–6) and these various preparations exhibited reduced-CO difference spectra with absorbance maxima in the region of 445 nm, a property shared with the members of the cytochrome P450 family. However, the low degree of sequence similarity to any of the more than 200 members of this family and other CO-binding, oxygenating heme proteins, such as chloroperoxidase or Bacillus megaterium P450 (BM-3), suggests that the homology may end with the sharing of a cysteine thiolate ligand to the heme iron at the fifth axial position. Recent studies have established that this involves cysteine415 of the neuronal NOS (11, 12), as suggested originally by McMillan, et al. (3), and cysteine184 of the endothelial NOS (13) as determined by site-directed mutagenesis of the holoenzyme (11,13) and the heme domain (12), respectively.

Inhibition of purified and membrane-bound flavin-containing monooxygenase 1 by (N,N-dimethylamino)stilbene carboxylates.

(E)-[2-(4-(Dimethylamino)phenyl)vinyl]benzenes bearing a nitrile or carboxyl group in the 2', 3', or 4' position were synthesized and tested for substrate activity with purified pig liver flavin-containing monooxygenase (FMO1). Although the nitrile derivatives were too insoluble to saturate the catalytic site at pH 7.4, they appeared to be substrates with K(m)'s somewhat above their maximum solubility (approximately equal to 0.1 mM) in the assay medium. Of the three carboxylic acid analogs, (E)-4-[2-(4(dimethylamino)phenyl)vinyl]benzoic acid had no detectable water solubility at pH 7.4, and measurements were restricted to (E)-3-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS3CO) and (E)-2-[2-(4-(dimethylamino)phenyl)vinyl]benzoic acid (DS2CO). While DS3CO and DS2CO were substrates, they also inhibited FMO1 turnover. DS3CO was the more effective inhibitor, and at 2 mM it inhibited FMO1 and microsomal-catalyzed oxidation of methimazole (N-methyl-2-mercaptoimidazole) by 80-90%. Kinetic studies indicated that the aminostilbene carboxylates were noncompetitive with both the xenobiotic substrate, methimazole, and NADPH. However, inhibition constants calculated from double reciprocal plots of velocity vs NADPH were K(i)(comp) 130 and 150 microM for DS3CO and DS2CO, respectively, whereas the uncompetitive Ki's were 10-15 times higher, which suggests that inhibition of NADPH binding may be primarily responsible for inhibition of FMO1 by the aminostilbene carboxylates. This model is also consistent with inhibition of cyclohexanone monooxygenase, a bacterial analog of FMO. DS3CO and DS2CO were again noncompetitive with methimazole but primarily competitive with NADPH. The aminostilbene carboxylates had no detectable effects on activity of pig or rat liver NADPH-cytochrome P450 reductase, which suggests that they are not nonspecific flavoprotein antagonists.

Species differences in 5α-androstane-3β,17β-diol hydroxylation by rat, monkey, and human prostate microsomes

The 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes appears to be catalyzed by a single, high-affinity cytochrome P450 enzyme. In the present study we have examined the hydroxylation of 5α-androstane-3β,17β-diol by prostate microsomes from cynomolgus monkeys and from normal subjects and patients with benign prostatic hyperplasia. Our results suggest that although rat, monkey, and human prostate microsomes catalyze the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol, these pathways of oxidation in monkeys and humans are not catalyzed by a single cytochrome P450 enzyme. The ratio of the three metabolites was not uniform among prostate microsomal samples from individual humans or monkeys. The 6α-hydroxylation of 5α-androstane-3β,17β-diol varied independently of both the 7α- and 7β-hydroxylation, which varied in unison. The 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by monkey prostate microsomes appeared to be differentially affected by in vivo treatment of monkeys with β-naphthoflavone or dexamethasone. Treatment of a monkey with dexamethasone appeared to cause a 2.5-fold increase in both the 7α- and the 7β-hydroxylation of 5α-androstane-3β,17β-diol without increasing the 6α-hydroxylation. The 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by human and monkey prostate microsomes, but not the 6α-hydroxylation, was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase. Similarly, the 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by human prostate microsomes, but not the 6α-hydroxylation, was markedly inhibited (>85%) by equimolar concentrations of the imidazole-containing antimycotic drugs ketoconazole, clotrimazole, and miconazole. These results suggest that the 7α- and 7β-hydroxylation of 5α-androstane-3β,17β-diol by monkey and human prostate microsomes is catalyzed by a cytochrome P450 enzyme, whereas the 6α-hydroxylation is catalyzed by a different enzyme which may or may not be a cytochrome P450 monooxygenase. The hydroxylation of 5α-androstane-3β,17β-diol by prostate microsomes from normal human subjects was quantitatively and qualitatively similar to its hydroxylation by prostate microsomes from patients with benign prostatic hyperplasia. This suggests that an impairment of androgen inactivation is not the mechanism for the proposed accumulation of 5α-dihydrotestosterone in the hyperplastic prostate. The rate of 5α-androstane-3β,17β-diol hydroxylation by human prostate microsomes ranged from 1 to 10 pmol/mg protein/min, which is considerably less than the rate catalyzed by rat prostate microsomes (~600 pmol/mg protein/min). This marked species difference in the rate of inactivation of androgens by prostate microsomes could potentially explain why humans, in contrast to rats, are prone to develop benign prostatic hyperplasia.

Hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes: Effects of antibodies and chemical inhibitors of cytochrome P450 enzymes

The purpose of the present study was to test the hypothesis that rat prostate microsomes contain a single cytochrome P450 enzyme responsible for the conversion of 5α-androstane-3β,17β-diol to a series of trihydroxylated products. The three major metabolites formed by in vitro incubation of 5α-[ 3H]androstane-3β,17β-diol with rat prostate microsomes were apparently 5α-androstane-3β,6α,17β-triol, 5α-androstane-3β,7α,17β-triol, and 5α-androstane-3β,7β,17β-triol, which were resolved and quantified by reverse-phase HPLC with a flow through radioactivity detector. The ratio of the three metabolites remained constant as a function of incubation time, microsomal protein concentration, ionic strength, and substrate concentration. The ratio of the three metabolites was dependent on pH, apparently because the hydroxylation of 5α-androstane-3β,17β-diol shifted from the 6α-to the 7α-position with increasing pH (6.8–8.0). The V max values were 380, 160, and 60 pmol/mg microsomal protein/min for the rate of 6α-, 7α-, and 7β-hydroxylation, respectively. Similar K m values (0.5–0.7 μ m) were measured for enzymatic formation of all three metabolites, which suggests that formation of all three metabolites was catalyzed by a single, high-affinity enzyme. Testosterone, 5α-dihydrotestosterone, and 5α-androstane-3α,17β-diol did not appreciably inhibit the hydroxylation of 5α-androstane-3β,17β-diol, suggesting that this enzyme exhibits a high degree of substrate specificity. Formation of all three metabolites was inhibited by antibody against rat liver NADPH-cytochrome P450 reductase (85%) and by a 9:1 mixture of carbon monoxide and oxygen (60%). Several chemical inhibitors of cytochrome P450 enzymes, especially the antimycotic drug clotrimazole, also inhibited the formation of all three metabolites. Polyclonal antibodies that recognize liver cytochrome P450 1A, 2A, 2B, 2C, and 3A enzymes did not inhibit 5α-androstane-3β,17β-diol hydroxylase activity. Overall, these results are consistent with the hypothesis that the 6α-, 7α-, and 7β-hydroxylation of 5α-androstane-3β,17β-diol by rat prostate microsomes is catalyzed by a single, high-affinity P450 enzyme. This cytochrome P450 enzyme appears to be structurally distinct from those in the 1A, 2A, 2B, 2C, and 3A gene families.

Spin-labeling studies of rat liver NADPH-cytochrome P450 reductase: Conformation and function relationship

ESR spin-labeling studies designed to yield information regarding the relationship between function and conformation of rat liver NADPH-cytochrome P450 reductase (EC 1.6.4.2) were carried out. The purified enzyme was spin labeled by a nitroxide derivative of p-chloromercuribenzoate. Two conditions for spin labeling were employed: (i) the presence of NADP +, yielding an active site-protected spin-labeled reductase, and (ii) the absence of NADP +, yielding completely spin-labeled reductase. Reductase in which the active site was protected by binding NADP + and then spin-labeled retains most of its enzymatic activity; on the other hand, completely spin-labeled reductase is devoid of any enzymatic activity. Completely spin-labeled reductase yields a two-component resolved ESR spectrum that reflects two classes of spin-labeled binding sites, a strongly immobilized (S) and a weakly immobilized (W) site. The ratio of W S provides a valuable parameter for studying the relationship between function and conformation. Structural perturbants, such as urea, KCl, and pH, were employed to determine their effects on the activity of the enzyme and their relationship to changes in the conformational state of the reductase. It was further observed that the enzymatically active spin-labeled derivative generated superoxide radical in the presence of NADPH and cytochrome c, which in turn reduced completely the attached spin-label.

Highly homologous cytochromes P-450 and b5: a model to study protein-protein interactions in a reconstituted monooxygenase system.

Cytochrome b5 from mouse and rat liver formed a type I spectral complex with two murine cytochrome P-450 isozymes, the P450Coh and P450PBI. Mouse b5 stimulated the reactions catalyzed by reconstituted P450Coh and an equimolar amount of b5 to P450Coh was needed for maximal effect. In contrast, rat b5 inhibited P450Coh-mediated reactions progressively starting from 1:1 ratio of b5 to P-450. Neither b5 had any effect on reactions catalyzed by P45015 alpha, an isozyme highly homologous to P450Coh, but with a point mutation (Arg-129----Ser) at site considered important for P-450-b5 interactions. In case of P450PBI, neither b5 protein had any effect on the associated activities at b5: P-450 ratios below 1, and a progressive inhibition occurred when b5: P-450 ratio was above 1. The results were similar with either rat or mouse liver NADPH-cytochrome P-450 reductase used in reconstitution demonstrating that the critical differences take place in P-450-b5 interactions. Kinetic and spectral experiments revealed that the stimulatory and inhibitory effects of b5 on the enzymatic reactions were due to corresponding changes in the reaction velocity, and that b5 does not compete with the flavoprotein nor with the substrate for binding to P-450. These results indicate that the high spin shift of P-450 does not necessarily correlate with enhanced reaction rates. Also, the increase in the coupling efficiency of P450PBI may result from the increased affinity for substrate in the presence of b5. Sequenation of mouse b5 peptides generated with proteinases revealed three amino acid changes between the mouse and rat b5, two of which appeared at the hydrophobic domain necessary for the P-450-b5 interaction. This could explain the species specificity of b5 proteins in supporting the P-450-mediated reactions. This is the first time functionally important differences in the interaction of highly homologous cytochromes P-450 and b5 have been demonstrated. Isozymes P45015 alpha and P450Coh, and mouse and rat b5 could serve as an excellent model for further studies on the nature and significance of P-450-b5 interactions.

Identification and characterization of an NADPH-cytochrome P450 reductase derived peptide involved in binding to cytochrome P450

The amino acids of cytochrome P450 reductase involved in the interaction with cytochrome P450 were identified with a differential labeling technique. The water-soluble carbodiimide EDC (1-ethyl-3-[3- (dimethylamino)propyl]-carbodiimide) was used with the nucleophile methylamine to modify carboxyl residues. When the modification was performed in the presence of cytochrome P450, there was no inhibition in the ability of the modified reductase to bind to cytochrome P450. However, subsequent modification of the reductase in the absence of cytochrome P450 caused a fourfold increase in the K m and an 80% decrease in k cat K m (relative to the reductase modified in the first step), for the interaction with cytochrome P450. These effects are attributed to the modification of approximately 3.2 mol of carboxyl residues per mole of reductase. Tryptic peptides generated from the modified reductase were purified by reverse phase high-performance liquid chromatography and characterized. Amino acid sequencing and analysis suggest that the peptide which contains approximately 40% of the labeled carboxyl residues corresponds to amino acid residues 109–130 of rat liver NADPH-cytochrome P450 reductase. One or more of the seven carboxyl containing amino acids within this peptide is presumably involved in the interaction with cytochrome P450.

Expression and enzymatic activity of recombinant cytochrome P450 17 alpha-hydroxylase in Escherichia coli.

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.

Characterization of multiple forms of cytochrome P-450 from an insecticide resistant strain of house fly ( Musca domestica)

Components of the microsomal monooxygenase system derived from abdominal preparations of an insecticide resistant house fly ( Musca domestica, Rutgers strain) were characterized. Cytochrome P-450 isozymes with molecular weights of 49,000, 53,000, 54,000, and 58,000 were partially purified using a procedure involving chromatography on columns of phenyl Sepharose, DEAE-cellulose (DE-52), carboxymethyl-Sepharose, hydroxylapatite, and an affinity column prepared from rat liver cytochrome b 5. Activity toward a variety of pesticides, as well as other exogenous and endogenous monooxygenase substrates, was examined using intact microsomes or purified isozymes in reconstituted systems. Oxidation of benzo[ a]pyrene in microsomal preparations was high although neither O-dealkylation of 7-ethoxyresorufin nor N-dealkylation of p-chloromethylaniline could be detected. Other substrates metabolized by abdominal microsomes from Rutgers flies included testosterone, lauric acid (primarily at in-chain positions), phorate, p-nitroanisole, and aldrin. Activity of abdominal microsomes from the susceptible CSMA strain toward benzo[ a]pyrene and lauric acid was, surprisingly, higher than that from the resistant Rutgers strain. Synergism between NADPH and NADH was noted with microsomes from either strain. Reconstitution experiments were relatively unsuccessful but activity toward lauric acid was obtained using a system that included purified cytochrome P-450 fractions, purified house fly or rat liver NADPH-cytochrome P-450 reductase, an NADPH generating system, and a phospholipid. Unlike similar systems utilizing mammalian cytochrome P-450, phosphatidylethanolamine was more active than phosphatidylcholine.

Aryl hydrocarbon hydroxylase activity in rat submandibular glands.

The activities of aryl hydrocarbon hydroxylase and 7-ethoxycoumarin O-de-ethylase in the rat submandibular gland, both of which are normally very low, were increased about 10- and 25-fold, respectively, by administration of 3-methylcholanthrene. The increased activity of aryl hydrocarbon hydroxylase was inhibited by 7,8-benzoflavone but little if any by metyrapone and SKF-525A. The submandibular gland aryl hydrocarbon hydroxylase was also inhibited by antibodies elicited against rat liver cytochrome P-448 and NADPH-cytochrome P-450 reductase, respectively, and slightly by anti-cytochrome b 5 antibody, suggesting that submandibular gland aryl hydrocarbon hydroxylase resembles that of the liver, not only in the immunochemical properties of cytochrome P-450 involved, but also in the entire enzyme complex, including the electron transferring system. Cofactor(s) required by aryl hydrocarbon hydroxylase in submandibular gland was NADPH, whereas NADH sustained the reaction at a much slower rate (about 20%). NADH showed some additive action when added with NADPH.

Purification of hepatic microsomal cytochromes P-450 from β-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 β-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 β-naphthoflavone. Both cytochrome P-450c and a minor form called cytochrome P-450d (56 000) 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 ( M r 55 000) or cytochrome P-450d in Ouchterlony immunodiffusion, but gave a precipitin line of partial identify with cod cytochrome P-450b ( M r 54 000), possibly as a result of contaminating cytochrome P-450c in this fraction.

Purification and properties of cytochrome P-450 from untreated monkey liver microsomes

Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogenous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448 1 and MC P-448 2) catalyzed 7-ethoxycoumarin O-deethylation activities.

Characterization and endocrine regulation of the cytochrome P-450 dependent microsomal hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol in the rat ventral prostate.

Of 11 structurally related steroids, 5 alpha-androstane-3 beta,17 beta-diol was the one most efficiently hydroxylated to polar metabolites by microsomes isolated from the rat ventral prostate. The metabolites were identified by gas chromatography-mass spectrometry as 6 alpha-,7 alpha-, and presumably 6 beta-hydroxylated 5 alpha-androstane-3 beta,17 beta-diol. The hydroxylated metabolites were separated and quantitated by high performance liquid chromatography using an aminopropylsilane hypersil column. Maximum velocity (V max) values of 0.36, 0.12, and 0.044 nmol/min X mg of microsomal protein were determined for the 6 alpha-,7 alpha-, and 6 beta-hydroxylase activities, respectively, with an apparent Michaelis-Menten constant (Km) of 30 microM for all hydroxylases. Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol in prostatic microsomes was NADPH dependent and was inhibited by several cytochrome P-450 inhibitors in vitro and by antibodies against rat liver NADPH-cytochrome P-450 reductase. The enzyme activities were stimulated by treatment of the rats with phenobarbital, beta-naphthoflavone, 5 alpha-androstane-3 beta,17 beta-diol,5 alpha-dihydrotestosterone, and methyltrienolone, whereas the activities were suppressed by ovine PRL, human GH, estradiol benzoate, cholesterol, andrenalectomy , hypophysectomy, and castration. It is concluded that the prostatic 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activities represent a constitutive cytochrome P-450 form in the rat ventral prostate with a high steroid substrate specificity. Endocrine factors are involved in the regulation of the activity of the enzyme. The ratio between the three hydroxylated metabolites was constant in almost all experiments suggesting that only one cytochrome P-450 species was involved in the hydroxylation.

Presence of NADPH-cytochrome P450 reductase in central catecholaminergic neurones.

The cytochrome P450-containing mixed function oxidases metabolize a variety of endogenous and exogenous compounds including drugs, carcinogens, fatty acids and steroids. Mixed function oxidases have been detected in several tissues, including brain. The enzyme system consists of a lipid fraction (phosphatidylcholine), cytochrome P450 and NADPH-cytochrome P450 reductase. NADPH-cytochrome P450 reductase has been purified to apparent homogeneity and demonstrated to supply reducing equivalents from NADPH to cytochrome P450 (refs 5-7). Detection of NADPH-cytochrome P450 reductase thus represents an indirect means of demonstrating the presence of cytochrome P450. Although the role of cytochrome P450 in the central nervous system (CNS) is not known, it may include such different functions as metabolism of xenobiotics, aromatization of androgens to oestrogens and the formation of catecholoestrogens. Despite the potentially very important role(s) of cytochrome P450 in brain function, its exact regional distribution remains essentially unknown. Using a specific antibody against rat liver NADPH-cytochrome P450 reductase in combination with immunohistochemical techniques, we have now localized this enzyme to define catecholamine (CA)-containing structures of the rat and monkey brain.

Cleavage of newly translated NADPH-cytochrome P-450 reductase in a rabbit reticulocyte lysate cell-free system in the absence of exogenous membranes

In a rabbit reticulocyte lysate cell-free system and using double antibody immunoprecipitation method, newly translated rat liver NADPH-cytochrome P-450 reductase (E.C. 1.6.2.4) was shown to be cleaved in the absence of exogenous membranes. Reductase having a M r=78,000 was shown to be converted to a M r=67,000 upon incubation with either lysate or antisera. Peptide maps of 78,000 dalton reductase and 67,000 dalton protein were identical except for three additional peptides present in the 78,000 dalton reductase map. The cleavage activity associated with the antisera could be prevented by using the IgG fraction, while that associated with the lysate was inhibited by using the protease inhibitors leupeptin, pepstatin or bestatin.

Immunological comparison of rat, rabbit, and human liver NADPH-cytochrome P-450 reductases.

NADPH-cytochrome P-450 reductase (EC 1.6.2.4) preparations were purified to electrophoretic homogeneity from rat, rabbit, and human liver microsomes. These preparations had apparent monomer molecular weights (Mr's) of 72 000-74 000 and were catalytically active in reducing rat and rabbit liver cytochromes P-450 as well as cytochrome c. A form of the human liver reductase devoid of a peptide of about Mr 6000 was isolated in the absence of protease inhibitors; this enzyme catalyzed the reduction of cytochrome c but not cytochromes P-450. Rabbits were immunized with purified rat liver NADPH-cytochrome P-450 reductase and the resulting antibody preparation was used to examine the species specificity of the enzyme. Immunological differences among the three species were detected by using double-diffusion analysis, quantitative microcomplement fixation, and inhibition of enzyme activity. Microcomplement fixation techniques indicated immunological differences in both rat and human reductase preparations due to removal of a peptide of Mr 6000-8000; these differences were not detected by using double-diffusion analysis. The antibody inhibited rat liver microsomal d-benzphetamine N-demethylase activity to the same extent as NADPh-cytochrome c reductase activity, suggesting that the level of reductase controls the rate of this cytochrome P-450-mediated activity. On the other hand, the antibody was much less effective in inhibiting rat liver benzo[a]pyrene hydroxylase activity. The antibody exerted different effects in inhibiting d-benzphetamine N-demethylase and benzo[a]pyrene hydroxylase activities as compared to NADPH-cytochrome c reductase activity in human liver microsomes.

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.