Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.     

Studies of cytochrome P-450 in testis microsomes

Studies on the role of cytochrome P-450 in mouse, rat, and chick testis microsomes showed that this CO-binding hemoprotein is involved in the activity of the 17α-hydroxylase. A 70–80% inhibition by CO of the 17α-hydroxylase activity was detected in rat and chick testis microsomes. In the mouse testis, the level of the enzyme activity is ten times greater than that of the rat. This partly explains why an acceleration of NADPH oxidation by progesterone can be observed in mouse but not in rat testis microsomes. In rat testis microsomes, type I binding spectra of cytochrome P-450 was observed with pregnenolone, progesterone, 17-hydroxyprogesterone, androstenedione, and testosterone. The apparent K_s values for progesterone and 17-hydroxyprogesterone were 0.50 and 1.00 μm, respectively.When NADPH is used to measure cytochrome P-450 levels in rat testis microsomes, CO formation resulting from a stimulation in lipid peroxidation by phosphate or Fe²⁺ was sufficient to bind with 50% of the total amount of cytochrome P-450. Substitution of phosphate by Tris reduced the amount of lipid peroxidation to minimal levels. On a comparable basis, no CO formation was observed in avian testis microsomes.An increase in the testicular levels of cytochrome P-450 resulted upon the administration of HCG and cyclic-AMP to 1-day-old chicks. The lack of stimulation of the cytochrome P-450 levels by progesterone and pregnenolone suggest that the hormonal stimulation of the P-450 levels is not due to substrate induction.     

Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: involvement of cytochrome P-450

Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide).     

Selective reactivation of steroid hydroxylases following dissociation of the isosafrole metabolite complex with rat hepatic cytochrome P-450

In order to elucidate the isozyme specificity of complex formation between cytochrome P-450 and the isosafrole metabolite the effect of complex dissociation on different steroid hydroxylation pathways was studied in hepatic microsomal fractions. Isosafrole induction was found to increase the 16 beta- and 7 alpha-hydroxylation of androst-4-ene-3,17-dione approximately 2.8- and 1.7-fold, respectively, whereas the 16 alpha-hydroxylation pathway was decreased to about one-quarter of control activity; 6 beta-hydroxylation was unchanged from control activity. More striking changes were apparent following dissociation of the isosafrole metabolite from its complex with ferricytochrome P-450 by the steroid substrate. Thus an approximate fourfold elevation of 16 beta-hydroxylase activity was observed after displacement and 6 beta-hydroxylation increased about twofold; 7 alpha-hydroxylase activity was decreased to 0.75-fold of undisplaced activity and 16 alpha-hydroxylase activity was unchanged. These data provide convincing evidence that at least two forms of phenobarbital-inducible cytochrome P-450 (cytochromes P-450PB-B and P-450PB/PCN-E) are present to some extent in a catalytically inactive complexed state in isosafrole-induced rat hepatic microsomes. Furthermore, there is now evidence to suggest that the constitutive isozymes cytochrome P-450UT-A and cytochrome P-450UT-F are not complexed to any degree in hepatic microsomes from isosafrole-induced rats.     

Theophylline metabolism by rat liver microsomal monooxygenases

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

Cytochrome P-450 dependent metabolism of testosterone in rat skin

The incubation of microsomes of whole skin, dermis and epidermis with 14C testosterone in the presence of NADPH resulted in the formation of 6 beta-, 7 alpha- and 16 alpha-testosterone. Maximum enzyme activity occurred in epidermal microsomes followed by dermis and whole skin. Epidermal testosterone hydroxylase activity required NADPH and oxygen and was found to be inhibited by SKF 525A and metyrapone. Our data strongly suggest that testosterone is metabolized by the cytochrome P-450 dependent monooxygenase in skin and provides the first evidence for an endogenous substrate for cytochrome P-450 in this tissue. The formation of several hydroxylated products further suggests the existence of multiple isozymes of cytochrome P-450 in rat skin. These studies provide additional evidence that target tissues may modulate their hormone levels by enzyme pathways that are locally regulated.     

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.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Cytochrome P-450 and halothane metabolism. Decrease in rat liver microsomal P-450 in vitro

Anaerobic in vitro incubation of microsomes from phenobarbital(PB)-induced rats with halothane results in an irreversible decrease of measurable cytochrome P-450. There is a parallel decrease in heme content under the same incubation conditions. However, microsomes from 3-methylcholanthrene(3-MC)-induced or untreated animals do not show a reduction in cytochrome P-450 content. Aerobic incubation with halothane results in a decrease of cytochrome P-450 which can be completely reversed by dialysis or the addition of potassium ferricyanide. These latter treatments only partially restore the cytochrome P-450 levels following anaerobic incubations. The decrease in cytochrome caused by halothane is not associated with measureable heme N-alkyl adduct formation; lipid peroxidation does not play a role as indicated by the lack of effect of 1 mM EDTA or a decrease in glucose-6-phosphatase activity. Halothane metabolites are bound irreversibly to microsomal protein as determined by gel electrophoresis only when the oxygen concentration is very low. The mechanism of cytochrome P-450 decrease is consistent with the formation of a reactive metabolite which binds to the protein portion and also destroys heme.     

Circulating C-terminal propeptide of type I procollagen is cleared mainly via the mannose receptor in liver endothelial cells.

The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-450(25] from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253, 549-552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol.mg of protein-1, and the protein showed a single spot with an apparent isoelectric point of 7.4 and an Mr of 50,500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124 pmol.min-1.nmol of cytochrome P-450-1 and towards 1 alpha-hydroxyvitamin D3 it was 1375 pmol.min-1.nmol-1. The preparation also catalysed the 25-hydroxylation of 5 beta-cholestane-3 alpha,7 alpha-diol at a rate of 1000 pmol.min-1.nmol of cytochrome P-450-1 and omega-1 hydroxylation of lauric acid at a rate of 200 pmol.min-1.nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-450(25) from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5 beta-cholestane-3 alpha,7 alpha-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-450(25) was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-450(25) from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450.     

Relation between the 7-ethoxyresorufin-O-deethylase activity of the liver microsomal monooxygenase system and the level of methylcholanthrene-induced form of cytochrome P-450

Changes in the metabolic activity of 7-ethoxyresorufin in rat liver microsomes containing different amounts of cytochrome P-450 induced by 3-methylcholanthrene and other polycyclic hydrocarbons (P-450c) were studied. Using antibodies to cytochrome P-450c for the determination of the cytochrome P-450c content and its metabolic role, it was demonstrated that 7-ethoxyresorufin O-deethylation by the liver microsomal monooxygenase system is catalyzed exclusively by cytochrome P-450c. The rate of the substrate metabolism is correlated with the cytochrome P-450c content in microsomal membranes; the cytochrome P-450c activity does not depend on the cytochrome P-450c/NADPH-cytochrome P-450 reductase ratio. The experimental results suggest that the level of 7-ethoxyresorufin metabolism in liver microsomes can be regarded as a measure of the cytochrome P-450c content, whose function is associated with the stimulation of potential carcinogenic and toxic substances.     

The role of cytochrome b5 in adrenal microsomal steroidogenesis.

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.     

Purification and characterization of cholesterol 7 alpha-hydroxylase cytochrome P-450 of untreated rabbit liver microsomes

Cytochrome P-450 which catalyzes the 7 alpha-hydroxylation of cholesterol was purified from liver microsomes of untreated rabbits. The minimum molecular weight of the cytochrome P-450 was estimated to be 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation contained 7 nmol of cytochrome per mg of protein. The oxidized form of the P-450 showed absorption maxima at 568, 535, and 417 nm, which are characteristic of a low spin hemoprotein, while the reduced form showed maxima at 545 and 413 nm. The carbon monoxide complex of the reduced form showed maxima at 550 and 447 nm. The cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes was reconstituted with the purified P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5. The P-450 catalyzed the 7 alpha-hydroxylation of cholesterol 500 times more efficiently than the starting microsomes. The reconstituted hydroxylase system showed a substantial salt dependency. In the presence of cytochrome b5 the activity was maximum at 0.4 M KCl (4.55 nmol product formed/mg of protein per min), whereas in the absence of cytochrome b5 the activity was marginal (0.65 nmol product formed/mg of protein per min) and inhibited by KCl. Thus, cytochrome b5 stimulated the hydroxylase activity by one order of magnitude. These results indicate that cytochrome b5 is an essential component of the cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes.     

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.     

Multiple forms of cytochrome P-450 in rabbit colon microsomes

Three cytochrome P-450 preparations, designated as cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction, were separated and purified about 23-, 50-, and 29-fold, respectively, from the cholate extracts of rabbit colon mucosa microsomes. Their specific contents were 1.2, 2.6, and 1.5 nmol of cytochrome P-450 per mg of protein, respectively. Cytochrome P-450ca and cytochrome P-450cb migrated as heme-containing polypeptide bands with molecular weights of about 53,000 and 57,000, respectively, on SDS-polyacrylamide gel electrophoresis. The CO-reduced difference spectra of cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction showed maxima at 451, 450, and 449 nm, respectively. Cytochrome P-450ca efficiently catalyzed the omega-hydroxylation of prostaglandin A1 (PGA1) and the omega- and (omega-1)-hydroxylation of caprate, laurate, and myristate in the reconstituted system containing cytochrome P-450ca, NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. In contrast, cytochrome P-450cb and cytochrome P-448c fraction had no detectable activity toward PGA1 and fatty acids. Both catalyzed aminopyrine and benzphetamine N-demethylation. Cytochrome P-448c fraction also hydroxylated benzo(a)pyrene, and phosphatidylinositol or phosphatidylserine exhibited a stimulatory effect on this activity. The results show that rabbit colon microsomes contain catalytically different cytochrome P-450, one of which is specialized for the omega-oxidation prostaglandins, the others being involved in the metabolism of exogenous compounds such as drugs and polycyclic hydrocarbons.     

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.     

Immunochemical studies on the metabolism of nitrosamines by ethanol-inducible cytochrome P-450

The ethanol-induced rabbit liver microsomal cytochrome P-450, P-450LM3a, has been shown previously to efficiently catalyze the demethylation of N-nitrosodimethylamine (NDMA) with a Km of 2.9 mM. Since the predominant Km in hepatic microsomes from ethanol-treated rabbits is 0.07 mM, the role of P-450LM3a in the activation of this carcinogen has been uncertain. In the present study, antibodies to P-450LM3a were shown to almost completely inhibit NDMA demethylation by the purified P-450 in a reconstituted system as well as the low-Km activity of liver microsomes from control or ethanol-treated rabbits. In contrast, the antibody did not inhibit the high-Km NDMA demethylase activity in the microsomes. These results indicate that P-450LM3a is the major P-450 responsible for the low-Km NDMA demethylase activity. In addition, evidence is provided for the existence of a cytochrome immunochemically similar to P-450LM3a in liver microsomes from rats, mice, and guinea pigs that effectively catalyzes the demethylation of NDMA.     

The influence of in vitro procedures which diminish NADPH cytochrome c reductase activity on oxidative drug metabolism in lung and liver microsomes

Rabbit lung and liver microsomes were subjected to three procedures which decreased NADPH cytochrome c reductase activity; flavoprotein antibody, trypsin and subtilisin digestion. The effects on benzphetamine and p-nitroanisole demethylation and amine metabolic-intermediate complex formation were investigated. In general, the proteolytic digestion had a greater inhibitory effect on oxidation reactions for a given loss of NADPH cytochrome c reductase activity than did flavoprotein antibody; and of the two proteases, subtilisin, which also diminishes the cytochrome b5 reduction pathway, had a greater inhibitory effect than trypsin. Subtilisin digestion had similar effects in both liver and lung microsomes; a loss of flavoprotein without a loss of cytochrome P-450; but whereas all three oxidative reactions decreased in unison as the flavoprotein was lost in the liver, benzphetamine demethylation was less susceptible to flavoprotein depletion than the other two reactions in lung microsomes. With trypsin digestion flavoprotein was removed without loss of cytochrome P-450 only in lung microsomes; in liver microsomes the cytochrome P-450 was susceptible to tryptic degradation. In lung microsomes, benzphetamine and p-nitroanisole demethylations were less susceptible to flavoprotein loss than metabolic-intermediate complex formation.