Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.14.13.97 (CYP3A4)
 6,365 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human cytochrome CYP3A4 is the most abundant of all the P450s in human liver and is involved in the metabolism of many environmental toxicants and drugs. Kinetic studies with CYP3A4 have been hampered due to low activity of this enzyme obtained from recombinant gene expression systems or difficulty in reconstituting activity with the native enzyme purified from human liver. To overcome these obstacles, we have expressed high levels of catalytically active CYP3A4 and human NADPH-cytochrome P450 reductase (CYPOR) together in two insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (T.ni), via a single recombinant baculovirus carrying both cDNAs (CYP3A4-OR). Microsomes containing recombinant CYP3A4-OR from these cell lines were up to 50-times more active in testosterone 6 beta-hydroxylase activity than recombinant CYP3A4 expressed alone and supplemented with purified rabbit CYPOR. The spectral P450 content of CYP3A4-OR T.ni microsomes was 107 pmol/mg microsomal protein and the cytochrome c reductase activity was 3904 units/mg. Recombinant CYP3A4-OR was catalytically similar to human liver CYP3A4 based on similarities in the testosterone metabolite profile, time course of metabolite formation, Vmax and Km values (for CYP3A4-OR, Vmax was 8.8 nmol/min/mg microsomal protein [70 nmol/min/nmol CYP3A4] and Km was 33 microM), the extent of inhibition by 100 microM troleandomycin (> 75%) in the presence of 25 microM testosterone, and the degree of P450 activation in the presence of 20 microM 7,8-benzoflavone. The coexpression of recombinant cytochrome b5 with CYP3A4-OR did not result in an additional increase in CYP3A4-OR activity.
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PMID:CYP3A4 expressed by insect cells infected with a recombinant baculovirus containing both CYP3A4 and human NADPH-cytochrome P450 reductase is catalytically similar to human liver microsomal CYP3A4. 777 80

To assess whether CYP3A4 and CYP3A7 have a similar capacity to activate carcinogenic mycotoxins, we established cell lines stably expressing human CYP3A4 and CYP3A7, which are adult- and fetal-specific forms of cytochrome P450 in human livers, respectively. Each cDNA was introduced into CR-119 cells which had been established by introducing guinea pig NADPH-cytochrome P450 reductase cDNA into Chinese hamster lung cells. The cell lines (4-line and 7-line) stably expressed the mRNA and the protein corresponding to CYP3A4 and CYP3A7, respectively. The concentration-response for aflatoxin B1 (AFB1) cytotoxicity in 4-line and 7-line, respectively, was compared. 4-10 and 7-40 cells were approximately 17- and 20 times more sensitive to AFB1 than the parental CR-119 cells, respectively. In addition, the sensitivities to AFB1 of both 4-10 and 7-40 cells were enhanced approximately seven times by the addition of 10 microM alpha-naphthoflavone, a known activator of CYP3A enzyme, while the sensitivities were suppressed approximately four times by the addition of 100 microM troleandomycin, which forms a metabolite intermediate complex with CYP3A enzyme. Moreover, both cell lines showed approximately 10 and 2 times higher sensitivity to sterigmatocystin and aflatoxin G1 than CR-119 cells, respectively. These results indicate that CYP3A4 and CYP3A7 have essentially similar capacities to activate AFB1, sterigmatocystin, and aflatoxin G1 to produce toxic metabolites.
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PMID:Fetus-specific CYP3A7 and adult-specific CYP3A4 expressed in Chinese hamster CHL cells have similar capacity to activate carcinogenic mycotoxins. 785 Jul 90

The present study investigated the role of rat and human cytochrome P450 enzymes in the sulfoxidation of S-methyl N,N-diethylthiolcarbamate (DETC-Me) to S-methyl N,N-diathylthiolcarbamate sulfoxide (DETC-Me sulfoxide), the putative active metabolite of disulfiram. DETC-Me sulfoxidation by microsomes from male and female rats treated with various cytochrome P450-enzyme inducers suggested that multiple enzymes can catalyze this reaction, and these include, CYP1A1/2, CYP2B1/2, and CYP3A1/2. All cDNA-expressed human cytochrome P450 enzymes examined catalyzed the sulfoxidation of DETC-Me. The turnover rates (min-1) of DETC-Me sulfoxidation by the cDNA-expressed cytochrome P450 enzymes ranked as follows: CYP3A4 > CYP2A6 = CYP2C9 > CYP1A2 > CYP2B6 = CYP2E1 > CYP1A1 > CYP2D6. Interestingly, CYP3A4 ranked first or last, depending on whether or not additional NADPH-cytochrome P450 reductase was coexpressed in the lymphoblastoid cells. This complicated estimates of the contribution of CYP3A4 to DETC-Me sulfoxidation by human liver microsomes. The sample-to-sample variation in DETC-Me sulfoxidation by bank of human liver microsomes (N=13) correlated highly with coumarin 7-hydroxylation (r=0.88) and testosterone 6beta-hydroxylation (r=0.90), suggesting that CYP2A6 and CYP3A4/5 contribute to the sulfoxidation of DETC-Me by human liver microsomes. Although, chlorzoxazone 6-hydroxylation (a marker for CYP2E1) correlated poorly with DETC-Me sulfoxidation, the correlation improved from r=0.07 to r=0.44 when DETC-Me sulfoxidation was studied in the presence of the CYP2A6 inhibitor, coumarin. Similarly, when DETC-Me sulfoxidation was studied in the presence of diethyldithiocarbamate (DDTC), the inhibited DETC-Me sulfoxidase activity correlated better (r=0.50) with chlorzoxazone 6-hydroxylase, compared with DETC-Me sulfoxidase activity in the absence of DDTC (r=0.09). Polyclonal antibodies against CYP2E1 caused a modest inhibition (30%) of DETC-Me sulfoxidation by human liver microsomes. Anti-CYP3A1 antibodies completely inhibited DETC-Me sulfoxidation by cDNA-expressed CYP3A4. Under similar conditions, DETC-Me sulfoxidation by human liver microsomes was only partially inhibited by anti-CYP3A1 antibodies. Although studies with the rat and cDNA-expressed cytochrome P450 enzymes suggested that CYP1A2 contributed to DETC-Me sulfoxidation, this reaction was not inhibited by either furafylline ( a mechanism-based inhibitor of CYP1A2) or antibodies against CYP1A1/2. A significant role for CYP2C9 was excluded by the inability of sulfaphenazole to inhibit the sulfoxidation of DETC-Me by human liver microsomes. Collectively, these data suggest that multiple cytochrome P450 enzymes can catalyze the sulfoxidation of DETC-Me. In human liver microsomes the CYP2A6, CYP2E1, and CYP3A4/5 all contribute significantly to the sulfoxidation of DETC-Me. It is interesting to note that DDTC, the reduced metabolite of disulfiram, is known to inhibit these same enzymes. The ability of DDTC to block the formation of DETC-Me sulfoxide may explain why the dose of disulfiram required to produce a disulfiram-ethanol reaction in alcoholics is so variable and often inadequate.
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PMID:Identification of the human and rat P450 enzymes responsible for the sulfoxidation of S-methyl N,N-diethylthiolcarbamate (DETC-ME). The terminal step in the bioactivation of disulfiram. 865 5

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.
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PMID:Effects of an immunosuppressive agent, tacrolimus (FK-506), on the activities of cytochrome P-450-linked monooxygenase systems in rat liver microsomes. 930 7

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.
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PMID:Role of human liver P450s and cytochrome b5 in the reductive metabolism of 3'-azido-3'-deoxythymidine (AZT) to 3'-amino-3'-deoxythymidine. 958 47

Native human cytochrome P4503A4 was most active in nifedipine oxidation when incorporated into a binary phospholipid vesicular system with human NADPH-cytochrome P450 reductase. The turnover numbers were estimated to be 17.6 and 19.6 min-1 in the presence of Mg2+ or Ca2+ ions (5 mmol/l) in the test system, respectively. Inclusion of b5 in the vesicular CYP3A4: reductase system results in a slightly lower nifedipine oxidase activity of 16.9 min-1 in the presence of Mg2+ ions. These results demonstrate that b5 is not an essential component in CYP3A4 catalyzed nifedipine oxidation in human liver.
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PMID:Investigations on the role of cytochrome b5 and divalent cations in the maximal nifedipine oxidase activity of human liver. 1036 11

Glutathione S-transferases and the cytochrome P450 system have been proposed for the vascular biotransformation systems in the metabolic activation of organic nitrates. The present study was designed to elucidate the role of human cytochrome P450 isoforms on nitric oxide formation from organic nitrates using lymphoblast microsomes transfected with human CYP isoforms cDNA. CYP3A4-transfected microsomes had the most effective potential of nitric oxide formation from isosorbide dinitrate. Anti-CYP3A2 antibody (which cross-reacts with CYP3A4) or ketoconazole (an inhibitor of the CYP3A superfamily) inhibited nitric oxide formation from isosorbide dinitrate in rat heart microsomes. Immunohistochemistry of human heart also showed intense bindings of CYP3A4 antibody in the endothelium of the endocardium and coronary vessels. These results suggest that the CYP3A4-NADPH-cytochrome P450 reductase system specifically participates in nitric oxide formation from isosorbide dinitrate.
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PMID:Isoforms of cytochrome P450 on organic nitrate-derived nitric oxide release in human heart vessels. 1038 83

LMC2 is the most abundant constitutively expressed hepatic cytochrome P450 found in sexually immature rainbow trout (Onchorynchus mykiss) and is also the isozyme that activates the carcinogen aflatoxin B1 (AFB1). This P450 has been cloned, sequenced, and designated as CYP2K1. The present report describes the heterologous expression of enzymatically active CYP2K1 (BV-CYP2K1) in baculovirus Spodoptera frugiperda (Sf9) insect cells and its catalytic and immunoreactivity characterization in comparison with that of the previously purified LMC2 P450. Homogenates of Sf9 cells expressing the CYP2K1 enzyme and LMC2 both catalyzed the hydroxylation of lauric acid and the epoxidation of AFB1 in the presence of rat NADPH-cytochrome P450 reductase. Both LMC2 and BV-CYP2K1 catalyzed the oxidation of lauric acid primarily at the (omega-1) position plus small amounts at the (omega-2) position. Formation of AFB1 epoxide was shown indirectly by the appearance of an AFB1 epoxide-glutathione conjugate when P450 incubation mixtures contained AFB1, glutathione (GSH) together with mouse liver cytosol or purified rat GSH-transferase. When the AFB1 epoxide-GSH conjugate produced by BV-CYP2K1 and purified LMC2 was analyzed by HPLC using a chiral column, it had a retention time identical to that produced by CYP3A4, a human P450 known to form exclusively the AFB1 exo-epoxide. These results, therefore, confirm that the cDNA-expressed CYP2K1 protein is catalytically and immunologically identical to purified trout LMC2 and that these two enzymes produce primarily the highly carcinogenic stereoisomeric exo-epoxide form of AFB1.
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PMID:Heterologous expression of CYP2K1 and identification of the expressed protein (BV-CYP2K1) as lauric acid (omega-1)-hydroxylase and aflatoxin B1 exo-epoxidase. 1103 53

To establish a prediction system for drug-induced gynecomastia in clinical fields, a model reaction system was developed to explain numerically this side effect. The principle is based on the assumption that 50% inhibition concentration (IC(50)) of drugs on the in vitro metabolism of estradiol (E2) to its major product 2-hydroxyestradiol (2-OH-E2) can be regarded as the index for achieving this purpose. By using human cytochrome P450s coexpressed with human NADPH-cytochrome P450 reductase in Escherichia coli as the enzyme, the reaction was examined. Among the nine enzymes (CYP1A1, 1A2, 2A6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) tested, CYP3A4 having a V(max)/K(m) (ml/min/nmol P450) value of 0.32 for production of 2-OH-E2 was shown to be the most suitable enzyme as the reagent. The inhibitory effects of ketoconazole, cyclosporin A, and cimetidine toward the 2-hydroxylation of E2 catalyzed by CYP3A4 were obtained, and their IC(50) values were 7 nM, 64 nM, and 290 microM, respectively. The present results suggest that IC(50) values thus obtained can be substituted as the prediction index for gynecomastia induced by drugs, considering the patients' individual information.
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PMID:Studies on the interactions between drugs and estrogen: analytical method for prediction system of gynecomastia induced by drugs on the inhibitory metabolism of estradiol using Escherichia coli coexpressing human CYP3A4 with human NADPH-cytochrome P450 reductase. 1106 38

The effect of changes in the N-terminal membrane-binding domain of cytochrome P450 forms and NADPH-cytochrome P450 reductase types on the cytochrome P450-dependent monooxygenase activities, has been examined. The nifedipine oxidase activity of two human P450 forms (CYP3A4, CYP3A4NF14) which differ only in their primary structure by ten amino acid residues in the N-terminal membrane-binding domain, yields nearly the same catalytic cycle time tau =2.65 +/- 0.15 s, due to their identical cytosolic catalytic protein structure. In contrast, the complex formation process ([P450]+[reductase] <--> [complex]) described by the dissociation constant KD, at high substrate concentration ([S]>>KS) and low product concentration ([P]<<KP) is determined to be KD/[P4501]o = 0.3 and 2.0, respectively. These values reflect large differences in the affinity of both P450 forms for the same type of reductase which is only due to their modified membrane-binding domains. In the present work, it has been shown for the first time, that the membrane-binding domain of cytochrome P450 enzymes determines the complexation process of the binary P450:reductase system. Furthermore, the nifedipine oxidase activity of the human CYP3A4 form reconstituted with two different types of reductase from human and rabbit also has the same catalytic cycle time tau = 2.65 +/- 0.15 s. This result is based on the similarity of the primary structure of the cytosolic catalytic domain of both reductase types. However, the complex was formed with different dissociation constants of KD/[P450]o = 0.3 and 4.7, respectively. This different affinity of both reductase types to the same P450 form is interpreted as a consequence of the substantial alteration of the amino acids in the N-terminal primary structure of their membrane-binding domains. 7-Ethoxycoumarin O-deethylase activity of two rat P450 forms (CYP2B1 and CYP1A1) were reconstituted with the same rat reductase. The catalytic cycle time for each P450 form is tau = 1.8 and 0.6 s, respectively. Correspondingly, the complex formation process controlled by the dissociation constant KD has changed from KD/[P450]o = 2.3 to 1.7, respectively. This is because both forms differ in their cytosolic as well as in their membrane-binding domains.
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PMID:Complexation of membrane-bound enzyme systems. 1109 25


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