CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase

The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.     

Identification of CYP4A11 as the major lauric acid omega-hydroxylase in human liver microsomes

Human liver microsomes are capable of oxidizing lauric acid (laurate), a model medium-chain fatty acid, at both the omega- and omega-1 positions to form 12- and 11-hydroxylaurate, respectively. These laurate hydroxylation reactions are apparently catalyzed by distinct P450 enzymes. While the P450 responsible for microsomal laurate omega-1 hydroxylation in human liver has been identified as CYP2E1, the enzyme catalyzing omega-hydroxylation remains poorly defined. To that end, we employed conventional purification and immunochemical techniques to characterize the major hepatic laurate omega-hydroxylase in humans. Western blotting with rat CYP4A1 antibodies was used to monitor a cross-reactive P450 protein (M(r) = 52 kDa) during its isolation from human liver microsomes. The purified enzyme (7.4 nmol P450/mg protein) had an NH2-terminal amino acid sequence identical to that predicted from the human CYP4A11 cDNA over the first 20 residues found. Upon reconstitution with P450 reductase and cytochrome b5, CYP4A11 proved to be a potent laurate omega-hydroxylase, exhibiting a turnover rate of 45.7 nmol 12-hydroxylaurate formed/min/nmol P450 (12-fold greater than intact microsomes), while catalyzing the omega-1 hydroxylation reaction at much lower rates (5.4 nmol 11-hydroxylaurate formed/min/nmol P450). Analysis of the laurate omega-hydroxylation reaction in human liver microsomes revealed kinetic parameters (a lone Km of 48.9 microM with a VMAX of 3.72 nmol 12-hydroxylaurate formed/min/nmol P450) consistent with catalysis by CYP4A11. In fact, incubation of human liver microsomes with antibodies raised to CYP4A11 resulted in nearly 85% inhibition of laurate omega-hydroxylase activity while omega-1 hydroxylase activity remained unaffected. Furthermore, a strong correlation (r = 0.89; P < 0.001) was found between immunochemically determined CYP4A11 content and laurate omega-hydroxylase activity in liver samples from 11 different subjects. From the foregoing, it appears that CYP4A11 is the principle laurate omega-hydroxylating enzyme expressed in human liver.     

Activation of the anti-cancer drug ifosphamide by rat liver microsomal P450 enzymes

The NADPH-dependent metabolism of ifosphamide catalyzed by rat liver microsomes was investigated in order to identify individual P450 enzymes that activate this anti-cancer drug and to ascertain their relationship to the P450 enzymes that activate the isomeric drug cyclophosphamide. Pretreatment of rats with phenobarbital or clofibrate increased by up to 8-fold the activation of both ifosphamide and cyclophosphamide catalyzed by isolated liver microsomes. Studies using P450 form-selective inhibitory antibodies demonstrated that constitutively expressed P450s belonging to subfamily 2C (forms 2C11/2C6) make significant contributions to the activation of both oxazaphosphorines in uninduced male rat liver microsomes, while the phenobarbital-inducible P450 2B1 was shown to be a major catalyst of these activations in phenobarbital-induced microsomes. Pretreatment of rats with dexamethasone increased liver microsomal activation of ifosphamide approximately 6-fold without a corresponding effect on cyclophosphamide activation rates. Ifosphamide activation catalyzed by dexamethasone-induced liver microsomes was minimally inhibited by anti-P450 2B or anti-P450 2C antibodies, but was selectively inhibited by anti-P450 3A antibodies. Selective inhibition of liver microsomal ifosphamide activation was also effected by the macrolide antibiotic triacetyloleandomycin, an inhibitor of several dexamethasone-inducible 3A P450s. These studies establish that a dexamethasone-inducible family 3A P450 can make an important contribution to rat liver microsomal ifosphamide activation, and suggest that dexamethasone pretreatment might provide a useful approach for modulation of ifosphamide metabolism in order to improve its therapeutic efficacy in cancer patients.     

Induction of cytochrome P450 1A in hamster liver and lung by 6-nitrochrysene

The present study has determined the effect of 6-nitrochrysene (6-NC) on hepatic and pulmonary cytochrome P450 (P450)-dependent monooxygenases using hamsters pretreated with the nitrated polycyclic aromatic hydrocarbon (nitro-PAH) at 5 mg/kg per day for 3 days. Pretreatment with 6-NC elevated serum gamma-glutamyltranspeptidase, lactate dehydrogenase, and bilirubin levels. Liver S9 fractions prepared from controls and hamsters pretreated with 6-NC markedly increased mutagenicity of the nitro-PAH in Salmonella typhimurium tester strains TA98, TA100, and TA102. The pretreatment selectively increased 1-nitropyrene reductase activities of lung cytosol and liver and lung microsomes. Pretreatment with 6-NC resulted in increases of microsomal 7-ethoxyresorufin and methoxyresorufin O-dealkylases activities in liver and lung without affecting the monooxygenase activities in kidney. Immunoblot analysis of microsomal proteins using mouse monoclonal antibody 1-12-3 to rat P450 1A1 revealed that 6-NC induced P450 1A-immunorelated proteins in liver and lung. RNA blot analysis using mouse P450 1A1 cDNA showed that 6-NC increased liver and lung P450 1A mRNA. 6-NC had no effect on the kidney P450 protein and mRNA. The present study demonstrates that the hamster enzymes can support 6-NC metabolic activation and the nitro-PAH induces liver and lung P4501A via a pretranslational mechanism.     

Inactivation of cytochrome P450 2E1 by tert-butylisothiocyanate

Several naturally occurring and synthethic isothiocyanates were evaluated for their ability to inactivate the major ethanol-inducible hepatic cytochrome P450 2E1. Of the compounds tested, tert-butylisothiocyanate (tBITC) was found to be the most selective inactivator of the 2E1 p-nitrophenol hydroxylation activity. tBITC was more specific for inactivating P450 2E1 activity than for rat P450 1A1, 1A2, 3A2, and 2B1, or the human cytochromes P450 3A4 and 2B6. The kinetics of inactivation of P450 2E1 by tBITC were characterized. P450 2E1, either in rat liver microsomes or in a purified reconstituted system containing the bacterially expressed rabbit cytochrome, was inactivated by tBITC in a mechanism-based manner. The loss of activity followed pseudo-first-order kinetics and was NADPH- and tBITC-dependent. The maximal rates for inactivation of P450 2E1 in microsomes or for the purified P450 2E1 at 30 degrees C were 0.72 and 0.27 min-1 and the apparent KI values were 11 and 7.6 microM, respectively. When cytochrome b5 was co-reconstituted with P450 2E1, the apparent KI for P450 2E1 inactivation by tBITC was similar to that seen in microsomes (14 microM). P450 2E1 T303A was also inactivated by tBITC with kinetic constants similar to that of the wild type enzyme. Co-incubations with an alternate substrate protected P450 2E1 from inactivation by tBITC. The extent of P450 2E1 inactivation by tBITC resulted in a comparable loss of the ability of the enzyme to form a reduced CO complex.     

Interactions of a bicyclic analog of colchicine with beta-tubulin isoforms alphabeta(II), alphabeta(III) and alphabeta(IV)

In this study we have investigated the occurrence of cytochrome P450 isoforms and of related cytochrome P450 reductase in human hepatic stellate cells (hHSC), a type of cell having relevant roles in physiopathological conditions of the liver. By performing immunoblotting of hHSC microsomes and immunofluorescence analysis associated to confocal laser microscopy we detected only P450 enzymes belonging to the cytochrome P450 3A subfamily (CYP3A) as well as cytochrome P450 reductase. The presence of CYP3A was further indicated by detection of testosterone 6beta-hydroxylase activity in hHSC microsomes. Other important human P450 forms were either undetectable (CYP1A2, CYP2E1, CYP2C8/9/19 and CYP4A) or bearly detectable (CYP1A1) in hHSC. This is the first study showing existence of active cytochrome P450 isoforms in human HSC.     

Effects of motorcycle exhaust on cytochrome P-450-dependent monooxygenases and glutathione S-transferase in rat tissues

The effects of motorcycle exhaust (ME) on cytochrome P-450 (P-450)-dependent monooxygenases were determined using rats exposed to the exhaust by either inhalation, intratracheal, or intraperitoneal administration. A 4-wk ME inhalation significantly increased benzo[a]pyrene hydroxylation, 7-ethoxyresorufin O-deethylation, and NADPH-cytochrome c reductase activities in liver, kidney, and lung microsomes. Intratracheal instillation of organic extracts of ME particulate (MEP) caused a dose- and time-dependent significant increase of monooxygenase activity. Intratracheal treatment with 0.1 g MEP extract/kg markedly elevated benzo[a]pyrene hydroxylation and 7-ethoxyresorufin O-deethylation activities in the rat tissues 24 h following treatment. Intraperitoneal treatment with 0.5 g MEP extract/kg/d for 4 d resulted in significant increases of P-450 and cytochrome b5 contents and NADPH-cytochrome c reductase activity in liver microsomes. The intraperitoneal treatment also markedly increased monooxygenases activities toward methoxyresorufin, aniline, benzphetamine, and erythromycin in liver and benzo[a]pyrene and 7-ethoxyresorufin in liver, kidney, and lung. Immunoblotting analyses of microsomal proteins using a mouse monoclonal antibody (Mab) 1-12-3 against rat P-450 1A1 revealed that ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment increased a P-450 1A protein in the hepatic and extrahepatic tissues. Protein blots analyzed using antibodies to P-450 enzymes showed that MEP intraperitoneal treatment caused increases of P-450 2B, 2E, and 3A subfamily proteins in the liver. The ME inhalation, MEP intratracheal, or MEP intraperitoneal treatment resulted in significant increases in glutathione S-transferase activity in liver cytosols. The present study shows that ME and MEP extract contain substances that can induce multiple forms of P-450 and glutathione S-transferase activity in the rat.     

Methodology of the survey]

To evaluate the effects of various barbiturates on lidocaine metabolism by cytochrome P-450 (P-450), enzyme kinetics were analyzed in an in vitro study using rat hepatic microsomes. Phenobarbital, amobarbital, hexobarbital, pentobarbital, and thiamylal showed the mixed type inhibition of lidocaine metabolism with inhibition constants being 4.89, 1.08, 2.76, 0.77 and 0.65 mM, respectively. Same as lidocaine, all barbiturates used in the present study, corresponding to binding with P-450, induced the I type of spectral change of P-450. Since these did not affect cytochrome C reductase activity, it was suggested that this inhibition of lidocaine metabolism in hepatic microsomes may have been caused by the reduction of activity on P-450 by the barbiturates.     

Some properties of a detergent-solubilized NADPH-cytochrome c(cytochrome P-450) reductase purified by biospecific affinity chromatography

NADPH-cytochrome c (cytochrome P-450) reductase (EC 1.6.2.4) has been purified to homogeneity, as judged by sodium dodecyl sulfate disc gel electrophoresis, from detergent-solubilized rat and pig liver microsomes using an affinity chromatography procedure. Treatment of microsomes with a polyethoxynonylphenyl ether plus either cholate or deoxycholate and subsequent batch-wise DEAE-cellulose chromatography followed by biospecific affinity chromatography on Sepharose 4B-bound N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate (2'5'-ADP-Sepharose 4B) result in a greater than 30% yield of purified reductase from microsomes. The enzyme contains 1 mol each of FAD and FMN and exhibits a molecular weight of 78,000 g mol-1 estimated by comparison with protein standards on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The turnover numbers calculated on the basis of flavin are 1360 min-1 and 1490 min-1 at 25 degrees for the pig and rat liver enzymes, respectively. Titration of these purified preparations aerobically with both NADPH and potassium ferricyanide demonstrated unequivocally that the air-stable, reduced form of NADPH-cytochrome c (P-450) reductase contains 2 electron equivalents, confirming recent results obtained by Masters et al. (Masters, B. S. S., Prough, R. A., and Kamin, H. (1975) Biochemistry 14, 607-613) for the proteolytically solubilized enzyme. In addition, these preparations are capable of reconstituting benzphetamine N-demethylation activity in the presence of partially purified cytochrome P-450 and dilauroylphosphatidylcholine, as measured by formaldehyde formation from benzphetamine.     

Absence of metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) by flavin-containing monooxygenase (FMO)

The N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a potent lung carcinogen present in tobacco and tobacco smoke. Carbonyl reduction, alpha-carbon hydroxylation (activation) and N-oxidation of the pyridyl ring (detoxification) are the three main pathways of metabolism of NNK. In this study, metabolism of NNK was studied with lung and liver microsomes from F344 rats, Syrian golden hamsters and pigs and cloned flavin-containing monooxygenases (FMOs) from human and rabbit liver. Thermal inactivation at 45 degrees C for 2 min reduced FMO S-oxygenating activity but did not affect N-oxidation of NNK, leading to the conclusion that FMOs are not implicated in the detoxification of NNK. Detoxification of NNK was not increased by n-octylamine or by incubation at pH 8.4, supporting the conclusion that FMOs are not involved in the metabolism of NNK. SKF-525A (1 mM) significantly reduced N-oxidation and alpha-carbon hydroxylation, suggesting that these two pathways were catalyzed by cytochromes P450. Metabolism of NNK was lower with lung microsomes than with liver microsomes. Inhibition of metabolism of NNK by SKF-525A was also observed with rat lung microsomes, leading to the conclusion that cytochromes P450 are involved in pulmonary metabolism of NNK. Cloned FMOs did not metabolize NNK. In conclusion, cytochromes P450 rather than FMOs are involved in N-oxidation of NNK. The high capacity of hamster liver microsomes to activate NNK does not correlate with the resistance of this tissue to NNK-induced hepatocarcinogenesis.     

Cloning of a human cDNA for CTP-phosphoethanolamine cytidylyltransferase by complementation in vivo of a yeast mutant

CTP-phosphoethanolamine cytidylyltransferase (ET) is the enzyme that catalyzes the formation of CDP-ethanolamine in the phosphatidylethanolamine biosynthetic pathway from ethanolamine. We constructed a Saccharomyces cerevisiae mutant of which the ECT1 gene, putatively encoding ET, was disrupted. This mutant showed a growth defect on ethanolamine-containing medium and a decrease of ET activity. A cDNA clone was isolated from a human glioblastoma cDNA expression library by complementation of the yeast mutant. Introduction of this cDNA into the yeast mutant clearly restored the formation of CDP-ethanolamine and phosphatidylethanolamine in cells. ET activity in transformants was higher than that in wild-type cells. The deduced protein sequence exhibited homology with the yeast, rat, and human CTP-phosphocholine cytidylyltransferases, as well as yeast ET. The cDNA gene product was expressed as a fusion with glutathione S-transferase in Escherichia coli and shown to have ET activity. These results clearly indicate that the cDNA obtained here encodes human ET.     

Involvement of human CYP1A isoenzymes in the metabolism and drug interactions of riluzole in vitro

Cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes involved in riluzole oxidation and glucuronidation were characterized in (1) kinetic studies with human hepatic microsomes and isoenzyme-selective probes and (2) metabolic studies with genetically expressed human CYP isoenzymes from transfected B-lymphoblastoid and yeast cells. In vitro incubation of [14C]riluzole (15 microM) with human hepatic microsomes and NADPH or UDPGA cofactors resulted in formation of N-hydroxyriluzole (K(m) = 30 microM) or an unidentified glucuroconjugate (K(m) = 118 microM). Human microsomal riluzole N-hydroxylation was most strongly inhibited by the CYP1A2 inhibitor alpha-naphthoflavone (IC50 = 0.42 microM). Human CYP1A2-expressing yeast microsomes generated N-hydroxyriluzole, whereas human CYP1A1-expressing yeast microsomes generated N-hydroxyriluzole, two additional hydroxylated derivatives and an O-dealkylated derivative. CYP1A2 was the only genetically expressed human P450 isoenzyme in B-lymphoblastoid microsomes to metabolize riluzole. Riluzole glucuronidation was inhibited most potently by propofol, a substrate for the human hepatic UGT HP4 (UGT1.8/9) isoenzyme. In vitro, human hepatic microsomal hydroxylation of riluzole (15 microM) was weakly inhibited by amitriptyline, diclofenac, diazepam, nicergoline, clomipramine, imipramine, quinine and enoxacin (IC50 approximately 200-500 microM) and cimetidine (IC50 = 940 microM). Riluzole (1 and 10 microM) produced a weak, concentration-dependent inhibition of CYP1A2 activity and showed competitive inhibition of methoxyresorufin O-demethylase. Thus, riluzole is predominantly metabolized by CYP1A2 in human hepatic microsomes to N-hydroxyriluzole; extrahepatic CYP1A1 can also be responsible for the formation of several other metabolites. Direct glucuronidation is a relatively minor metabolic route. In vivo, riluzole is unlikely to exhibit significant pharmacokinetic drug interaction with coadministered drugs that undergo phase I metabolism.     

Effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems in rat liver microsomes

The effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems with respect to three cytochrome P-450 isozymes in rat liver microsomes were investigated. FK-506 non-competitively inhibited the aniline p-hydroxylase, p-nitroanisole O-demethylase and lidocaine N-deethylase activities of cytochrome P-450-linked monooxygenase systems, these activities being mainly catalyzed by cytochromes P-450 CYP2E1, CYP2C11 and CYP3A4, respectively, and the Ki values of the activities for FK-506 were determined to be 605, 491 and 97 microM, respectively. The inhibition of cytochrome P-450-linked monooxygenase systems by FK-506 seemed to involve the direct inhibition of cytochromes P-450 because the NADPH-cytochrome c reductase and NADPH-ferricyanide reductase activities of NADPH-cytochrome P-450 reductase were not affected by the presence of 1 mM FK-506 at all. A spectrophotometric study showed that a reverse type I spectral change was induced on the addition of FK-506 to rat liver microsomes, and the Ks value was apparently 125 microM. On the other hand, the EPR spectra of cytochromes P-450 in rat liver microsomes were not affected by 1 mM FK-506. These results suggest direct interaction between FK-506 and cytochrome P-450 apoproteins, except for the heme iron regions of cytochromes P-450, resulting in inhibition of the drug-metabolism activities catalyzed by cytochromes P-450.     

Vitamin E in therapy of rheumatic diseases]

Methyl t-butyl ether (MTBE) and ethyl t-butyl ether (ETBE) are commonly used in unleaded gasoline to increase the oxygen content of fuel and to reduce carbon monoxide emissions from motor vehicles. This study was undertaken to investigate: (1) the effect of administration to rats of ETBE and its metabolite, t-butanol, on the induction and/or inhibition of hepatic P450 isoenzymes; (2) the oxidative metabolism of MTBE and ETBE by liver microsomes from rats pretreated with selected P450 inducers and purified rat P450(s), (2B1, 2E1, 2C11, 1A1). ETBE administration by gavage at a dose of 2 ml/kg for 2 days induced hepatic microsomal P4502E1-linked p-nitrophenol hydroxylase and the P4502B1/2-associated PROD and 16beta-testosterone hydroxylase, verified by immunoblot experiments. t-Butanol treatments at doses of 200 and 400 mg/kg i.p. for 4 days did not alter any liver microsomal monoxygenases. Both MTBE and ETBE were substrates for rat liver microsomes and were oxidatively dealkylated to yield formaldehyde and acetaldehyde, respectively. The dealkylation rates of both MTBE and ETBE were increased c. fourfold in phenobarbital (PB)-treated rats. In rats pretreated with pyrazole, an inducer of 2E1, only the demethylation of MTBE was increased (c. twofold). When the oxidations of MTBE and ETBE were investigated with purified P450(s) in a reconstituted system, it was found that P4502B1 had the highest activities towards both solvents, whereas 1A1 and 2C1 were only slightly active; P4502E1 had an appreciable activity on MTBE but not against ETBE. Metyrapone, a potent inhibitor of P450 2B, consistently inhibited both the MTBE and ETBE dealkylations in microsomes from PB-treated rats. Furthermore, 4-methylpyrazole (a probe inhibitor of 2E1) and anti-P4502E1 IgG showed inhibition, though modest, only on MTBE demethylation, but not on ETBE deethylation. Inhibition experiments have also suggested that rat 2A1 may exert an important role in MTBE and ETBE oxidation. Taken together, these results indicate that 2B1, when expressed, is the major enzyme involved in the oxidation of these two solvents and that 2E1 may have a role, although minor, in MTBE demethylation. The implications of these data for MTBE and ETBE toxicity remain to be established.     

Characterization of the n-alkane and fatty acid hydroxylating cytochrome P450 forms 52A3 and 52A4

Two enzymes, P450 52A3 (P450Cm1) and 52A4 (P450Cm2), the genes of which belong to the CYP52 multigene family occurring in the alkane-assimilating yeast Candida maltosa, have been characterized biochemically and compared in terms of their substrate specificities. For this purpose, both the p450 proteins and the corresponding C. maltosa NADPH-cytochrome P450 reductase were separately produced by expressing their cDNAs in Saccharomyces cerevisiae, purified, and reconstituted to active monooxygenase systems. Starting from microsomal fractions with a specific content of 0.75 nmol P450Cm1, 0.34 nmol P450Cm2, and 10.5 units reductase per milligram of protein, respectively, each individual recombinant protein was purified to homogeneity. P450 substrate difference spectra indicated strong type I spectral changes and high-affinity binding of n-hexadecane (Ks= 26 micron) and n-octadecane (Ks = 27 microM) to P450Cm1, whereas preferential binding to P450Cm2 was observed using lauric acid (Ks = 127 microM) and myristic acid (Ks = 134 microM) as substrates. These substrate selectivities were further substantiated by kinetic parameters, determined for n-alkane and fatty acid hydroxylation in a reconstituted system, which was composed of the purified components and phospholipid, as well as in microsomes obtained after coexpressing each of the P450 proteins with the reductase. The highest catalytic activities within the reconstituted system were measured for n-hexadecane hydroxylation to 1-hexadecanol by P450Cm1 (Vmax = 27 microM x min-1, Km = 54 microM) and oxidation of lauric acid to 16-hydroxylauric acid by P450Cm2 (Vmax = 30 microM x min-1, Km = 61 microM). We conclude that P450Cm1 and P450Cm2 exhibit overlapping but distinct substrate specificities due to different chain-length dependencies and preferences for either n-alkanes or fatty acids.     

Sequence of a mouse embryo cDNA clone encoding proteolipid subunit 9 (P1) of the mitochondrial H(+)-ATP synthase

A cDNA clone to an abundantly expressed mRNA in cleavage stage mouse embryos has been sequenced and identified as encoding subunit 9 (P1) of the mitochondrial H(+)-ATP synthase. The deduced amino acid sequence of the mature subunit 9 protein differs in a single residue from the corresponding rat, ovine, bovine and human subunits.     

Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine

Our laboratory has shown that human liver microsomes metabolize the anti-HIV drug 3'-azido-3'-deoxythymidine (AZT) via a P450-type reductive reaction to a toxic metabolite 3'-amino-3'-deoxythymidine (AMT). In the present study, we examined the role of specific human P450s and other microsomal enzymes in AZT reduction. Under anaerobic conditions in the presence of NADPH, human liver microsomes converted AZT to AMT with kinetics indicative of two enzymatic components, one with a low Km (58-74 microM) and Vmax (107-142 pmol AMT formed/min/mg protein) and the other with a high Km (4.33-5.88 mM) and Vmax (1804-2607 pmol AMT formed/min/mg). Involvement of a specific P450 enzyme in AZT reduction was not detected by using human P450 substrates and inhibitors. Antibodies to human CYP2E1, CYP3A4, CYP2C8, CYP2C9, CYP2C19, and CYP2A6 were also without effect on this reaction. NADH was as effective as NADPH in promoting microsomal AZT reduction, raising the possibility of cytochrome b5 (b5) involvement. Indeed, AZT reduction among six human liver samples correlated strongly with microsomal b5 content (r2 = 0.96) as well as with aggregate P450 content (r2 = 0.97). Upon reconstitution, human liver b5 plus NADH:b5 reductase and CYP2C9 plus NADPH:P450 reductase were both effective catalysts of AZT reduction, which was also supported when CYP2A6 or CYP2E1 was substituted for CYP2C9. Kinetic analysis revealed an AZT Km of 54 microM and Vmax of 301 pmol/min for b5 plus NADH:b5 reductase and an AZT Km of 103 microM and Vmax of 397 pmol/min for CYP2C9 plus NADPH:P450 reductase. Our results indicate that AZT reduction to AMT by human liver microsomes involves both b5 and P450 enzymes plus their corresponding reductases. The capacity of these proteins and b5 to reduce AZT may be a function of their heme prothestic groups.