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Hirani V Yarovoy A Kozeska A Magnusson RP Lasker JM 《Archives of biochemistry and biophysics》2008,478(1):59-68
P450 enzymes comprising the human CYP4F gene subfamily are catalysts of eicosanoid (e.g., 20-HETE and leukotriene B4) formation and degradation, although the role that individual CYP4F proteins play in these metabolic processes is not well defined. Thus, we developed antibodies to assess the tissue-specific expression and function of CYP4F2, one of four CYP4F P450s found in human liver and kidney. Peptide antibodies elicited in rabbits to CYP4F2 amino acid residues 61-74 (WGHQGMVNPTEEG) and 65-77 (GMVNPTEEGMRVL) recognized on immunoblots only CYP4F2 and not CYP4F3b, CYP4F11 or CYP4F12. Immunoquantitation with anti-CYP4F2 peptide IgG showed highly variable CYP4F2 expression in liver (16.4+/-18.6pmol/mg microsomal protein; n=29) and kidney cortex (3.9+/-3.8 pmol/mg; n=10), with two subjects lacking the hepatic or renal enzyme entirely. CYP4F2 content in liver microsomes was significantly correlated (r> or =0.63; p<0.05) with leukotriene B4 and arachidonate omega-hydroxylase activities, which are both CYP4F2-catalyzed. Our study provides the first example of a peptide antibody that recognizes a single CYP4F P450 expressed in human liver and kidney, namely CYP4F2. Immunoquantitation and correlation analyses performed with this antibody suggest that CYP4F2 functions as a predominant LTB4 and arachidonate omega-hydroxylase in human liver. 相似文献
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CYP4F1 was discovered by Chen and Hardwick (Arch. Biochem. Biophys. 300, 18-23, 1993) as a new CYP4 cytochrome P450 (P450) preferentially expressed in rat hepatomas. However, the catalytic function of this P450 remained poorly defined. We have purified recombinant CYP4F1 protein to a specific content of 12 nmol of P450/mg of protein from transfected yeast cells by chromatography of solubilized microsomes on an amino-n-hexyl Sepharose 4B column, followed by sequential HPLC on a DEAE column and two hydroxylapatite columns. The purified P450 was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 53 kDa. The enzyme catalyzed the omega-hydroxylation of leukotriene B(4) with a K(m) of 134 microM and a V(max) of 6.5 nmol/min/nmol of P450 in the presence of rabbit hepatic NADPH-P450 reductase and cytochrome b(5). In addition, 6-trans-LTB(4), lipoxin A(4), prostaglandin A(1), and several hydroxyeicosatetraenoic acids (HETEs) were also omega-hydroxylated. Of several eicosanoids examined, 8-HETE was the most efficient substrate, with a K(m) of 18.6 microM and a V(max) of 15.8 nmol/min/nmol of P450. In contrast, no activity was detected toward lipoxin B(4), laurate, palmitate, arachidonate, and benzphetamine. The results suggest that CYP4F1 participates in the hepatic inactivation of several bioactive eicosanoids. 相似文献
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Leukotriene B4 (LTB4) omega-hydroxylase activity in human neutrophil microsomes was significantly inhibited by antisera against three rabbit omega-hydroxylase P-450s, lung prostaglandin omega-hydroxylase (P-450p-2), small intestine prostaglandin A omega-hydroxylase (P-450ia), and kidney fatty acid omega-hydroxylase (P-450kd). In contrast, the activity is not affected by antibodies raised against the phenobarbital-inducible forms of P-450s from both rabbits and rats. These findings suggest that the LTB4 omega-hydroxylase (P-450LTB omega) is structurally related to a group of rabbit omega-hydroxylase P-450s. The antiserum raised against P-450p-2 also inhibited the NADPH-dependent oxidation of 20-hydroxy LTB4 to 20-oxo LTB4 and 20-carboxy LTB4 by the microsomes, supporting that P-450LTB omega is able to catalyze the subsequent oxidation of 20-hydroxy LTB4 as well as the omega-hydroxylation of LTB4. 相似文献
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Bylund J Harder AG Maier KG Roman RJ Harder DR 《Archives of biochemistry and biophysics》2003,412(1):34-41
Leukotriene B(4) (LTB(4)) is a lipid mediator that plays an important role in inflammation. Metabolism of LTB(4) by cytochrome P450 (CYP) enzymes belonging to the CYP4F subfamily is considered to be of importance for the regulation of inflammation. This study investigates LTB(4) metabolism by recombinant rat CYP4F5 and CYP4F6 expressed in a yeast system and by microsomes isolated from rat organs expressing CYP4F mRNA. CYP4F6 was found to convert LTB(4) into 19-hydoxy- and 18-hydroxy-LTB(4) with an apparent K(m) of 26 microM, and CYP4F5 was found to convert LTB(4) primarily into 18-hydroxy-LTB(4) with an apparent K(m) of 9.7 microM. The rate of formation of 18-hydroxy-LTB(4) by CYP4F5 was surprisingly high. At a substrate concentration of 30 microM, the rate of formation was about 15 nmol/min/mg microsomal protein, approximately 30 times faster than the reaction catalyzed by CYP4F6. Analysis of LTB(4) metabolism by microsomes isolated from various tissues from the rat suggests that CYP4F5 and CYP4F6 are active in the lung and to some extent in the brain, kidney, and testis. CYP4F5 and CYP4F6, due to their capacities to metabolize LTB(4), may play important roles in modulating inflammatory response in these organs. 相似文献
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CYP4F isoforms are involved in the oxidation of important cellular mediators such as leukotriene B4 (LTB4) and prostaglandins. The proinflammatory agent LTB4 and cytotoxic leukotoxins have been associated with several inflammatory diseases. We present evidence that the hydroxylation of Z 9(10)-epoxyoctadecanoic, Z 9(10)-epoxyoctadec-Z 12-enoic, and Z 12(13)-epoxyoctadec-Z 9-enoic acids and that of monoepoxides from arachidonic acid [epoxyeicosatrienoic acid (EET)] is important in the regulation of leukotoxin and EET activity. These three epoxidized derivatives from the C18 family (C18-epoxides) were converted to 18-hydroxy-C18-epoxides by human hepatic microsomes with apparent Km values of between 27.6 and 175 microM. Among recombinant P450 enzymes, CYP4F2 and CYP4F3B catalyzed mainly the omega-hydroxylation of C18-epoxides with an apparent Vmax of between 0.84 and 15.0 min(-1), whereas the apparent Vmax displayed by CYP4F3A, the isoform found in leukocytes, ranged from 3.0 to 21.2 min(-1). The rate of omega-hydroxylation by CYP4A11 was experimentally found to be between 0.3 and 2.7 min(-1). CYP4F2 and CYP4F3 exhibited preferences for omega-hydroxylation of Z 8(9)-EET, whereas human liver microsomes preferred Z 11(12)-EET and, to a lesser extent, Z 8(9)-EET. Moreover, vicinal diol from both C18-epoxides and EETs were omega-hydroxylated by liver microsomes and by CYP4F2 and CYP4F3. These data support the hypothesis that the human CYP4F subfamily is involved in the omega-hydroxylation of fatty acid epoxides. These findings demonstrate that another pathway besides conversion to vicinal diol or chain shortening by beta-oxidation exists for fatty acid epoxide inactivation. 相似文献
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Leukotriene B4 (LTB4), a potent chemotactic agent, was catabolized to 20-hydroxyleukotriene B4 (20-OH-LTB4) by the 150,000 x g pellet (microsomal fraction) of human neutrophil sonicate. The reaction required molecular oxygen and NADPH, and was significantly inhibited by carbon monoxide, suggesting that a cytochrome P-450 is involved. The neutrophil microsomal fraction showed a carbon monoxide difference spectrum with a peak at 450 nm in the presence of NADPH or dithionite, indicating the presence of a cytochrome P-450. The addition of LTB4 to the microsomal fraction gave a type-I spectral change with a peak at around 390 nm and a trough at 422 nm, indicating a direct interaction of LTB4 with the cytochrome P-450. The dissociation constant of LTB4, determined from the difference spectra, is 0.40 microM, in agreement with the kinetically determined apparent Km value for LTB4 (0.30 microM). Such a spectral change was not observed with prostaglandins A1, E1 and F2 alpha or lauric acid, none of which inhibited the LTB4 omega-hydroxylation. The inhibition of the LTB4 omega-hydroxylation by carbon monoxide was effectively reversed by irradiation with monochromatic light of 450 nm wavelength. The photochemical action spectrum of the light reversal of the inhibition corresponded remarkably well with the carbon monoxide difference spectrum. These observations provide direct evidence that the oxygen-activating component of the LTB4 omega-hydroxylase system is a cytochrome P-450. Ferricytochrome c inhibited the hydroxylation of LTB4 and the inhibition was fortified by cytochrome oxidase. An antibody raised against rat liver NADPH-cytochrome-P-450 reductase inhibited both LTB4 omega-hydroxylase activity and the NADPH-cytochrome-c reductase activity of human neutrophil microsomal fraction. These observations indicate that NADPH-cytochrome-P-450 reductase acts as an electron carrier in LTB4 omega-hydroxylase. On the other hand, an antibody raised against rat liver microsomal cytochrome b5 inhibited the NADH-cytochrome-c reductase activity but not the LTB4 omega-hydroxylase activity of human neutrophil microsomal fraction, suggesting that cytochrome b5 does not participate in the LTB4-hydroxylating system. These characteristics indicate that the isoenzyme of cytochrome P-450 in human neutrophils, LTB4 omega-hydroxylase, is different from the ones reported to be involved in omega-hydroxylation reactions of prostaglandins and fatty acids. 相似文献
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M C Romano R D Eckardt P E Bender T B Leonard K M Straub J F Newton 《The Journal of biological chemistry》1987,262(4):1590-1595
omega-Hydroxylation of leukotriene B4 (LTB4) has been reported in human and rodent polymorphonuclear leukocytes; preliminary information indicates that this metabolism is cytochrome P-450 dependent. Therefore, these studies were initiated to characterize the cytochrome P-450-dependent metabolism of LTB4 in other tissues. LTB4 was metabolized by rat hepatic microsomes to two products, 20-hydroxy(omega)-LTB4 and 19-hydroxy(omega-1)-LTB4. The formation of these metabolites was both oxygen and NADPH dependent indicating that a monooxygenase(s) was responsible for these reactions. The apparent Km and Vmax for LTB4 omega-hydroxylase were 40.28 microM and 1202 pmol/min/mg of protein, respectively. In contrast, the apparent Km and Vmax for LTB4 (omega-1)-hydroxylase were 61.52 microM and 73.50 pmol/min/mg of protein, respectively. Both LTB4 omega- and (omega-1)-hydroxylases were inhibited by metyrapone in a concentration-dependent fashion. However, SK&F 525A inhibited LTB4 (omega-1)- but not omega-hydroxylase. In contrast, alpha-naphthoflavone decreased LTB4 omega- but not (omega-1)-hydroxylase activities. The differences in the Km apparent for substrate as well as the differential inhibition by inhibitors of cytochrome P-450 suggest that the omega- and (omega-1)-hydroxylations of LTB4 in hepatic microsomes are mediated by different isozymes of P-450. Furthermore, several additional characteristics of LTB4 hydroxylases indicate that these isozymes of P-450 may be different from those which catalyze similar reactions on medium-chain fatty acids, such as laurate and prostaglandins. 相似文献
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The omega-hydroxylation of leukotriene B4 (LTB4) by rat liver microsomes requires NADPH and molecular oxygen, suggesting that the hydroxylation is catalyzed by a cytochrome P-450 (P-450)-linked monooxygenase system. The reaction is inhibited by CO, and the inhibition is reversed by irradiation of light at 450 nm in a light-intensity-dependent manner. The extent of the reversal is strongly dependent on the wavelength of the light used, the 450-nm light is most efficient. The finding provides direct evidence for the identification of the LTB4 omega-hydroxylase as a P-450. The P-450 seems to be also responsible for prostaglandin A1 (PGA1) omega-hydroxylation, but not for lauric acid omega-hydroxylation. The LTB4 omega-hydroxylation is competitively inhibited by PGA1, but not affected by lauric acid. The Ki value for PGA1 of 38 microM agrees with the Km value for PGA1 omega-hydroxylation of 40 microM. LTB4 inhibits the PGA1 omega-hydroxylation by rat liver microsomes in a competitive manner with the Ki of 43 microM, which is consistent with the Km for the LTB4 omega-hydroxylation of 42 microM. An antiserum raised against rabbit pulmonary PG omega-hydroxylase (P-450p-2) inhibits slightly the omega-hydroxylations of LTB4 and PGA1, while it has stronger inhibitory effect on lauric acid omega-hydroxylation. In addition to NADPH-cytochrome P-450 reductase, cytochrome b5 appears to participate in the LTB4 omega-hydroxylating system, since the reaction is inhibited by an antibody raised against the cytochrome b5 as well as one raised against the reductase. 相似文献
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Leukotriene B4 (LTB4), a potent chemoattractant for leukocytes, is catabolized by human neutrophils via omega-oxidation. Neutrophil microsomes are known to oxidize 20-hydroxy-LTB4 (20-OH-LTB4) to its 20-oxo and 20-carboxy derivatives in the presence of NADPH. This activity has been ascribed to LTB4 omega-hydroxylase (cytochrome P-450LTB omega), a conclusion supported by our finding of the reversal of carbon monoxide inhibition by 450 nm light and by competitive inhibition studies. The oxidation of 20-oxo-LTB4 to 20-carboxy-LTB4 is also catalyzed by microsomes fortified with 1 mM NAD+, and this activity is not affected by cytochrome P-450LTB omega inhibitors. The evidence is compatible with involvement of a disulfiram-insensitive aldehyde dehydrogenase in this second oxidation pathway. Interaction of the two pathways is evidenced by facilitation of NADPH-dependent oxidation of 20-OH-LTB4 by the addition of NAD+. This synergism may be explained by removal of the aldehyde intermediate by the NAD(+)-dependent aldehyde dehydrogenase. Taken together with the finding that the NAD(+)-dependent activity is severalfold higher than the NADPH-dependent one, the dehydrogenase may be important in the oxidation of 20-OH-LTB4 to 20-carboxy-LTB4. 相似文献
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Christmas P Tolentino K Primo V Berry KZ Murphy RC Chen M Lee DM Soberman RJ 《The Journal of biological chemistry》2006,281(11):7189-7196
Leukotriene B(4) (LTB(4)) is a potent chemoattractant for polymorphonuclear leukocytes (PMN) and other cells. Human PMN inactivate LTB(4) by omega-oxidation catalyzed by cytochrome P-450 (CYP) 4F3A. The contribution of the enzymatic inactivation of LTB(4) by CYP4Fs to down-regulating functional responses of cells to LTB(4) is unknown. To elucidate the role of CYP4F-mediated inactivation of LTB(4) in terminating the responses of PMN to LTB(4) and to identify a target for future genetic studies in mice, we have identified the enzyme that catalyzes the omega-1 and omega-2 oxidation of LTB(4) in mouse myeloid cells as CYP4F18. As determined by mass spectrometry, this enzyme catalyzes the conversion of LTB(4) to 19-OH LTB(4) and to a lesser extent 18-OH LTB(4). Inhibition of CYP4F18 resulted in a marked increase in calcium flux and a 220% increase in the chemotactic response of mouse PMN to LTB(4). CYP4F18 expression was induced in bone marrow-derived dendritic cells by bacterial lipopolysaccharide, a ligand for TLR4, and by poly(I.C), a ligand for TLR3. However, when bone marrow-derived myeloid dendritic cells trafficked to popliteal lymph nodes from paw pads, the expression of CYP4F18 was down-regulated. The results identify CYP4F18 as a critical protein in the regulation of LTB(4) metabolism and functional responses in mouse PMN and identify it as the functional orthologue of human PMN CYP4F3A. 相似文献
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The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 microM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6beta-hydroxylation (r2=0.9). There was also a strong correlation between 6alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6beta-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 microM concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 microM). This might give an explanation for the limited formation of 6alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver. 相似文献
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Hashizume T Imaoka S Hiroi T Terauchi Y Fujii T Miyazaki H Kamataki T Funae Y 《Biochemical and biophysical research communications》2001,280(4):1135-1141
A cDNA encoding a novel human CYP4F enzyme (designated CYP4F12) was cloned by PCR from a human small intestine cDNA library. RT-PCR analysis demonstrated that CYP4F12 is expressed in human small intestine and liver. This cDNA contains an entire coding region of a 524-amino-acid protein that is 81.7, 78.3, and 78.2% identical to CYP4F2, CYP4F3, and CYP4F8, respectively. When expressed in Saccharomyces cerevisiae, the P450 catalyzes leukotriene B(4) omega-hydroxylation and arachidonic acid omega-hydroxylation, typical reactions of CYP4F isoforms. Their activity levels are, however, much lower than those of CYP4F2. Interestingly, CYP4F12 catalyzes the hydroxylation of the antihistamine ebastine with significantly higher catalytic activity relative to CYP4F2 (385 vs 5 pmol/min/nmol P450). These results indicate that CYP4F12 has a different profile of substrate specificity from other CYP4F isoforms, enzymes responsible for metabolizing endogenous autacoids, therefore suggesting that it may play an important role in xenobiotic biotransformation in the human small intestine. 相似文献
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Antoun J Goulitquer S Amet Y Dreano Y Salaun JP Corcos L Plée-Gautier E 《Journal of lipid research》2008,49(10):2135-2141
The regulation of the human liver-specific cytochrome P450 4F3B (CYP4F3B) isoform, a splice variant of the CYP4F3 gene with strong substrate specificity for long chain fatty acids, is yet an unsolved question. This report provides the first evidence that CYP4F3B is uniquely induced by prostaglandin A(1) (PGA(1)) in human hepatocyte-like HepaRG cells and leads to the synthesis of 20-hydroxy-eicosatetraenoic acids (HETEs). Real time PCR, immunoblot analysis with a specific antipeptide antibody, and determination of fatty acid omega-hydroxylase activity demonstrate that PGA(1) treatment strongly increases expression of CYP4F3B. This induction drives the production of 20-HETE (19-fold increase). SiRNA-mediated-silencing of CYP4F3 suppresses both 20-HETE synthesis and PGA(1) induced 20-HETE production. Taken together, these results provide evidence that CYP4F3B is the key enzyme to produce 20-HETE by omega-hydroxylation of arachidonic acid in liver cells. Since 20-HETE is a potent activator of PPARalpha and an important inflammatory mediator, CYP4F3B may exert important functions in lipid homeostasis and in inflammatory diseases. 相似文献
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Leukotriene B4 metabolism by hepatic cytochrome P-450 总被引:2,自引:0,他引:2
Bernhard Bösterling James R. Trudell 《Biochemical and biophysical research communications》1983,114(2):850-854
Leukotriene B4 (LTB) was found to be metabolized by suspensions of rat liver microsomes in the presence of NADPH and oxygen. The rate of LTB metabolism was also measured in reconstituted systems of both micelles and phospholipid vesicles containing cytochrome P-450-LM2, NADPH cytochrome P-450 reductase, and cytochrome b5. A 1 microM concentration of LTB was metabolized by rat hepatic microsomes at a rate of 4 pmol LTB/min/nmole P-450, and by vesicle and micelle reconstituted systems at 3 pmole/min/nmole P-450-LM2. At this rate a 10 g rat liver exposed to 1 microM LTB can metabolize 30 micrograms per hour. In that the leukotrienes are pharmacologically active at nanomolar concentrations, hepatic metabolism may be an important pathway of leukotriene inactivation. 相似文献
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Catalytic activities of human liver cytochrome P-450 IIIA4 expressed in Saccharomyces cerevisiae 总被引:2,自引:0,他引:2
W R Brian M A Sari M Iwasaki T Shimada L S Kaminsky F P Guengerich 《Biochemistry》1990,29(51):11280-11292
A human liver cytochrome P-450 (P-450) IIIA4 cDNA clone was inserted behind an alcohol dehydrogenase promoter in the plasmid vector pAAH5 and expressed in Saccharomyces cerevisiae (D12 and AH22 strains). A cytochrome P-450 with typical spectral properties was expressed at a level of approximately 8 x 10(5) molecules/cell in either strain of yeast. The expressed P-450 IIIA4 had the same apparent monomeric Mr as the corresponding protein in human liver microsomes (P-450NF) and could be isolated from yeast microsomes. Catalytic activity of the yeast microsomes toward putative P-450 IIIA4 substrates was seen in the reactions supported by cumene hydroperoxide but was often lower and variable when supported by the physiological donor NADPH. The catalytic activity of purified P-450 IIIA4 was also poor in some systems reconstituted with rabbit liver NADPH-P-450 reductase and best when both the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and a lipid extract (from liver or yeast microsomes) or L-alpha-1,2-dilauroyl-sn-glycero-3-phosphocholine were present. Under these conditions the expressed P-450 IIIA4 was an efficient catalyst for nifedipine oxidation, 6 beta-hydroxylation of testosterone and cortisol, 2-hydroxylation of 17 beta-estradiol and 17 alpha-ethynylestradiol, N-oxygenation and 3-hydroxylation of quinidine, 16 alpha-hydroxylation of dehydroepiandrosterone 3-sulfate, erythromycin N-demethylation, the 10-hydroxylation of (R)-warfarin, the formation of 9,10-dehydrowarfarin from (S)-warfarin, and the activation of aflatoxins B1 and G1, sterigmatocystin, 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (both + and - diastereomers), 3,4-dihydroxy-3,4-dihydrobenz[a]anthracene, 3,4-dihydroxy-3,4-dihydro-7, 12-dimethylbenz[a]anthracene, 9,10-dihydroxy-9,10-dihydrobenzo[b]fluoranthene, 6-aminochrysene, and tris(2,3-dibromopropyl) phosphate to products genotoxic in a Salmonella typhimurium TA1535/pSK1002 system where a chimeric umuC' 'lacZ plasmid is responsive to DNA alkylation. Reaction rates were stimulated by 7,8-benzoflavone and inhibited by rabbit anti-P-450 IIIA (anti-P-450NF), troleandomycin, gestodene, and cimetidine. Evidence was obtained that rates of reduction of ferric P-450 IIIA4 in yeast microsomes and the reconstituted systems are slow and at least partially responsible for the lower rates of catalysis seen in these systems (relative to liver microsomes). The results of these studies with a defined protein clearly demonstrate the ability of P-450 IIIA4 to catalyze regio- and stereoselective oxidations with a diverse group of substrates, and this enzyme appears to be one of the most versatile catalysts in the P-450 family. 相似文献