[Analysis of nuclear matrix protein in human lung cancer cells].
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Adenocarcinoma↗
Biomedical subjects
H Yamazaki
Publications and source records attributed to H Yamazaki.
[A childhood case of systemic lupus erythematosus associated with myasthenia gravis].
We described a 12-year-old girl with systemic lupus erythematosus (SLE) associated with myasthenia gravis (MG). She had absence seizures from 6 years old. She admitted to our hospital at 12 years of age because of absence seizures and dyspnea. The diagnosis of SLE was made on the basis of convulsion, arthritis, pleurisy, and positive antinuclear factor and was started therapy with prednisolone. The clinical course was complicated by the appearance of dysphagia and hoarseness. On the basis of positive Tensilon test and a high level of serum anti-acetylcholine receptor antibody, we made a diagnosis of the systemic type of MG. Her condition was improved by methylprednisolone pulse therapy and gamma-globulin therapy after plasmapheresis. The association of early-onset SLE with MG is rare.
Age of Onset↗
[Effect on 5'-deoxy-5-fluorouridine (5'-DFUR) of pyrimidine nucleoside phosphorylase (PyNPase), matrix metalloprotease and serum IAP values. Hokuriku Colorectal Cancer Chemotherapy Study Group].
PyNPase activity, MMPs activity and serum IAP values were measured in tumor tissues from colorectal cancer patients who had been divided into two groups, one given preoperative 5'-DFUR and the controls. PyNPase activity of the preoperative administration group was approximately equivalent to that of the controls. In the control group, correlations were assessed between PyNPase activity and activities of MMP1 and MMP3. To assess the effect of 5'-DFUR on the activity of MMPs, we divided patients into two groups, a high and a low PyNPase activity group. Although there was no correlation with MMPs activity of the preoperative administration group and the control group in the low PyNPase activity group, the activities of MMP1 and MMP9 of the control group were significantly higher in the high PyNPase activity group. Moreover, the serum IAP value of the administration group was significantly lower than that of the control group. These results indicated that PyNPase activity was thus suggested to be somehow related to MMPs activity and serum IAP values.
Adult↗
Different contributions of cytochrome P450 2C19 and 3A4 in the oxidation of omeprazole by human liver microsomes: effects of contents of these two forms in individual human samples.
Omeprazole 5-hydroxylation and sulfoxidation activities were determined in liver microsomes of different humans whose levels of individual forms of cytochrome P450 (P450 or CYP) varied. Correlation coefficients between omeprazole 5-hydroxylation activities (when determined at a substrate concentration of 10 microM) and S-mephenytoin 4'-hydroxylation and testosterone 6beta-hydroxylation activities were found to be 0.64 and 0.67, respectively, in liver microsomes of 84 human samples examined. Omeprazole sulfoxidation activities in these human samples were correlated with testosterone 6beta-hydroxylation activities (r = 0. 86). Omeprazole 5-hydroxylation by liver microsomes of a human sample that contained relatively high levels of CYP3A4 and low levels of CYP2C19 were inhibited very significantly by ketoconazole and anti-CYP3A4 antibodies, although a human sample having high in CYP2C19 and low in CYP3A4 was found to be sensitive toward fluvoxamine and anti-CYP2C9 antibodies. Sulfaphenazole (at 100 microM) did not affect the omeprazole 5-hydroxylation and sulfoxidation catalyzed by human liver microsomes. Both recombinant human CYP2C19 and CYP3A4 enzymes had activities for omeprazole 5-hydroxylation, with low Km and high Vmax values for the former enzyme and high Km and low Vmax values for the CYP3A4. These results suggest that contributions of CYP2C19 and CYP3A4 in the omeprazole 5-hydroxylation depend upon the ratio of these two P450 levels in human liver microsomes. Omeprazole 5-hydroxylation activities of different human samples were found to be related to predicted values calculated from the kinetic parameters of recombinant enzymes and the levels of liver microsomal CYP2C19 and CYP3A4 enzymes. Finally, when recombinant human CYP2C19 and CYP3A4 were mixed at levels found in different human samples, relatively similar profiles of omeprazole oxidation by the recombinant and microsomal enzyme systems were determined by analysis of high-performance liquid chromatography. These results suggest that both CYP2C19 and CYP3A4 are involved in the 5-oxidation of omeprazole (at a substrate concentration of 10 microM) in human liver microsomes and that contributions of these P450 enzymes depend on the compositions of CYP2C19 and CYP3A4 in liver.
Animals↗
Lack of electron transfer from cytochrome b5 in stimulation of catalytic activities of cytochrome P450 3A4. Characterization of a reconstituted cytochrome P450 3A4/NADPH-cytochrome P450 reductase system and studies with apo-cytochrome b5.
Many catalytic activities of cytochrome P450 (P450) 3A4, the major human liver P450 enzyme, require cytochrome b5 (b5) for optimal rates. The stimulatory effect of b5 on P450 reactions has generally been thought to be due to transfer of electrons from ferrous b5 to the ferrous P450-O2-substrate complex. We found that apo-b5, devoid of heme, could substitute for b5 in stimulating two prototypic activities, testosterone 6beta hydroxylation and nifedipine oxidation. The stimulatory effect was not seen with albumin, hemoglobin, catalase, or cytochrome c. Apo-b5 could not substitute for b5 in a testosterone 6beta hydroxylation system composed of NADH-b5 reductase and P450 3A4. Rates of electron transfer from NADPH-P450 reductase to ferric P450 3A4 were too slow (<2 min-1) to support testosterone 6beta hydroxylation ( approximately 14 min-1) unless b5 or apo-b5 was present, when rates of approximately 700 min-1 were measured. The oxidation-reduction potential (Em,7) of the ferric/ferrous couple of P450 3A4 was unchanged ( approximately -310 mV) under different conditions in which the kinetics of reduction were altered by the addition of substrate and/or apo-b5. Rapid reduction of P450 3A4 required substrate and a preformed complex of P450 3A4, NADPH-P450 reductase, and b5; the rates of binding of the proteins to each other were 2-3 orders of magnitude less than reduction rates. We conclude that b5 can facilitate some P450 3A4-catalyzed oxidations by complexing with P450 3A4 and enhancing its reduction by NADPH-P450 reductase, without directly transferring electrons to P450.
Animals↗
Effect of FR167653, a cytokine suppressive agent, on endotoxin-induced disseminated intravascular coagulation.
FR167653 (1-[7-(4-fluorophenyl)-1,2,3,4-tetrahydro-8-(4-pyridyl)pyrazolo[5-1-c] [1,2,4]triazin-2-yl]-2-phenylethanedione sulfate monohydrate) is a low molecular weight inflammatory cytokine inhibitor that inhibits the production of interleukin-1 alpha, interleukin-1 beta and tumor necrosis factor-alpha (TNF-alpha) in human monocytes stimulated with lipopolysaccharide, and in human lymphocytes stimulated with phytohemagglutinin-M. FR167653 inhibited these cytokines in a dose-dependent manner (IC50 values were 0.84, 0.088, 1.1 microM and 0.072, respectively). However, FR167653 did not inhibit even at 10 microM interleukin-6 production by human monocytes, and the production of interleukin-2 and interferon-gamma by human lymphocytes. We evaluated the effect of FR167653 on lipopolysaccharide-induced disseminated intravascular coagulation in rats. FR167653 (0.032-0.32 mg/kg/h for 4 h, intravenous infusion) markedly improved thrombocytopenia and plasma coagulation parameters in a dose-dependent manner, but not leukopenia in this mode. Plasma interleukin-1 and TNF-alpha levels were elevated by lipopolysaccharide administration and the treatment with FR167653 (0.31 mg/kg/h for 4 h) inhibited the increased plasma interleukin-1 (100.0%) and plasma TNF-alpha (89.2%) levels. These results suggest that interleukin-1 and TNF-alpha may play a pivotal role in the pathogenesis of DIC. FR167653 can act as a protective drug in lipopolysaccharide-induced DIC, and this protection is due to an inhibition of increased plasma interleukin-1 and TNF-alpha.
Animals↗
Elements of neural adhesion molecules and a yeast vacuolar protein sorting receptor are present in a novel mammalian low density lipoprotein receptor family member.
Normal cell development depends to a large part on multifunctional proteins that have evolved by recombination of proven modular elements. We now have discovered and characterized in rabbit such a multi-domain protein, and classify it as novel member of the low density lipoprotein (LDL) receptor gene family. The extracellular portion of the approximately 250-kDa membrane protein, termed LR11, contains a cluster of 11 LDL receptor ligand binding repeats, a group of 5 LDL receptor "YWTD" repeats, a large hexarepeat domain of structural elements found in neural cell adhesion molecules, and a domain with similarity to a yeast receptor for vacuolar protein sorting, VPS10. The cytoplasmic domain exhibits features typical of endocytosis-competent coated-pit receptors. The mosaic, and presumably multifunctional, receptor is expressed abundantly in brain, in particular the hippocampus, dentate gyrus, and cerebral cortex, and is present at significant levels in liver, adrenal glands, and testis. Western blotting of tissues and ligand blotting of LR11-transfected cells demonstrated that the novel protein binds apolipoprotein E-containing lipoproteins. In contrast to the LDL receptor, hepatic expression of LR11 is unaffected by hyperlipidemia. The identification of this highly conserved and superbly complex protein offers the opportunity to gain new insights into the emergence of multifunctional mosaic proteins akin to the ever expanding LDL receptor gene family.
Amino Acid Sequence↗
Observation of S=1 fractional spins in the S=2 finite linear-chain Heisenberg antiferromagnet CsCr1-xMgxCl3.
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Journal Article↗
Cloning and characterization of KAP3: a novel kinesin superfamily-associated protein of KIF3A/3B.
We previously reported that KIF3A and KIF3B form a heterodimer that functions as a microtubule-based fast anterograde translocator of membranous organelles. We have also shown that this KIF3A/3B forms a complex with other associated polypeptides, named kinesin superfamily-associated protein 3 (KAP3). In the present study, we purified KAP3 protein by immunoprecipitation using anti-KIF3B antibody from mouse testis. Microsequencing was carried out, and we cloned the full-length KAP3 cDNA from a mouse brain cDNA library. Two isoforms of KAP3 exist [KAP3A (793 aa) and KAP3B (772 aa)], generated by alternative splicing in the carboxyl terminus region. Their amino acid sequences have no homology with those of any other known proteins, and prediction of their secondary structure indicated that almost the entire KAP3 molecule is alpha-helical. We produced recombinant KAP3 and KIF3A/3B using a baculovirus-Sf9 expression system. A reconstruction study in Sf9 cells revealed that KAP3 is a globular protein that binds to the tail domain of KIF3A/3B. The immunolocalization pattern of KAP3 was similar to that of KIF3A/3B in nerve cells. In addition, we found that KAP3 does not affect the motor activity of KIF3A/3B. KAP3 was associated with a membrane-bound form of KIF3A/3B in a fractional immunoprecipitation experiment, and since the KIF3 complex was found to bind to membranous organelles in an EM study, KAP3 may regulate membrane binding of the KIF3 complex.
Adaptor Proteins, Signal Transducing↗
Mapping rat megalin: the second cluster of ligand binding repeats contains a 46-amino acid pathogenic epitope involved in the formation of immune deposits in Heymann nephritis.
Megalin (gp330), an epithelial endocytic receptor, is a major target antigen of Heymann nephritis (HN), an autoimmune disease in rats. To elucidate the mechanisms of HN, we have mapped a pathogenic epitope in megalin that binds anti-megalin antibodies. We focused our attention on four clusters of cysteine-rich, low density lipoprotein receptor (LDLR) ligand binding repeats in the extracellular domain of megalin because they represent putative ligand binding regions and therefore would be expected to be exposed in vivo and to be able to bind circulating antibodies. Rat megalin cDNA fragments I through IV encoding the first through fourth clusters of ligand-binding repeats, respectively, were expressed in a baculovirus system. All four expression products were detected by immunoblotting with two antisera capable of inducing passive HN (pHN). When antibodies eluted from glomeruli of rats with pHN were used for immunoblotting, only the expression product encoded by fragment II was detected. This indicates that the second cluster of LDLR ligand binding repeats is directly involved in binding anti-megalin antibodies and in the induction of pHN. To narrow the major epitope in this domain, fragment II was used to prepare proteins sequentially truncated from the C- and N-terminal ends by in vitro translation. Analysis of the truncated translation products by immunoprecipitation with anti-megalin IgG revealed that the fifth ligand-binding repeat (amino acids 1160-1205) contains the major epitope recognized. This suggests that a 46-amino acid sequence in the second cluster of LDLR ligand binding repeats contains a major pathogenic epitope that plays a key role in pHN. Identification of this epitope will facilitate studies on the pathogenesis of HN.
Amino Acid Sequence↗
Requirements for cytochrome b5 in the oxidation of 7-ethoxycoumarin, chlorzoxazone, aniline, and N-nitrosodimethylamine by recombinant cytochrome P450 2E1 and by human liver microsomes.
NADH-dependent 7-ethoxycoumarin O-deethylation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant P450 2E1 in 100 mM potassium phosphate buffer (pH 7.4) containing a synthetic phospholipid mixture and cholate. Replacement of NADH-b5 reductase with NADPH-P450 reductase yielded a 4-fold increase in 7-ethoxycoumarin O-deethylation activity, and further stimulation (approximately 1.5-fold) could be obtained when NADPH was used as an electron donor. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of 7-ethoxycoumarin O-deethylation activities in the presence of NADPH-P450 reductase, but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. Km values were increased and Vmax values were decreased for 7-ethoxycoumarin O-deethylation when b5 was omitted from the NADPH-supported P450 2E1-reconstituted systems. Requirements for b5 in P450 2E1 systems were also observed in chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation. In human liver microsomes, NADH-dependent 7-ethoxycoumarin O-deethylation, chlorzoxazone 6-hydroxylation, aniline p-hydroxylation, and N-nitrosodimethylamine N-demethylation activities were found to be about 55, 41, 33, and 50%, respectively, of those catalyzed by NADPH-supported systems. Anti-rat NADPH-P450 reductase immunoglobulin G inhibited 7-ethoxycoumarin O-deethylation activity catalyzed by human liver microsomes more strongly in NADPH- than NADH-supported reactions, while anti-human b5 immunoglobulin G inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. These results suggest that b5 is an essential component in P450 2E1-catalyzed oxidations of several substrates used, that about 10% of the activities occur via P450 2E1 reduction by NADPH-P450 reductase in the absence of b5, and that the NADH-supported system contributes, in part, to some reactions catalyzed by P450 2E1 in human liver microsomes.
Aniline Compounds↗
Activation of chemically diverse procarcinogens by human cytochrome P-450 1B1.
A human cytochrome P-450 (P450) 1B1 cDNA was expressed in Saccharomyces cerevisiae and the microsomes containing P450 1B1 were used to examine the selectivity of this enzyme in the activation of a variety of environmental carcinogens and mutagens in Salmonella typhimurium TA1535/pSK1002 or NM2009 tester strains, using the SOS response as an end point of DNA damage. We also determined and compared these activities of P450 1B1 with those catalyzed by recombinant human P450s 1A1 and 1A2, which were purified from membranes of Escherichia coli. The carcinogenic chemicals tested included 27 polycyclic aromatic hydrocarbons and their dihydrodiol derivatives, 17 heterocyclic and aryl amines and aminoazo dyes, three mycotoxins, two nitroaromatic hydrocarbons, N-nitrosodimethylamine, vinyl carbamate, and acrylonitrile. Among the three P450 enzymes examined here, P450 lB1 was found to have the highest catalytic activities for the activation of 11,12-dihydroxy-11,12-dihydrodibenzo[a,l]pyrene, 1,2-dihydroxy-1,2-dihydro-5-methylchrysene, (+)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 11,12-dihydroxy-11,12-dihydrobenzo[g]chrysene, 3,4-dihydroxy-3,4-dihydrobenzo[c]phenanthrene, 3-amino-1,4-dimethyl-5H-pyrido[4,3-b]indole, 2-aminoanthracene, 3-methoxy-4-aminoazobenzene, and 2-nitropyrene. P450 1B1 also catalyzed the activation of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline, 2-amino-3-methylimidazo[4,5-f]quinoline, 2-aminofluorene, 6-aminochrysene and its 1,2-dihydrodiol, (-)-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene, 1,2-dihydroxy-1,2-dihydrochrysene, 1,2-dihydroxy-1,2-dihydro-5,6-dimethylchrysene, 2,3-dihydroxy-2,3-dihydrofluoranthene, 3,4-dihydroxy-3,4-dihydro-7,12-dimethylbenz[a]anthracene, and 6-nitrochrysene to appreciable extents. However, P450 1B1 did not produce genotoxic products from benzo[a]pyrene, trans- 3,4-dihydroxy-3,4-dihydrobenzo[a]anthracene, trans-8,9-dihydroxy-8,9-dihydrobenzo[a]anthracene, 7,12-dimethylbenz[a]anthracene and its cis-5,6-dihydrodiol, 5-methylchrysene, 11,12-dihydroxy-11,12-dihydro-3-methylcholanthrene, 1,2-dihydroxy-1,2-dihydro-6-methylchrysene, benzo[c]phenanthrene, 2-amino-6-methyldipyridol[1,2-a:3',2'-d]imidazole, 2-acetylaminofluorene, benzidine, 2-naphthylamine, aflatoxin B1, aflatoxin G1, sterigmatocystin, N-nitrosodimethylamine, vinyl carbamate, or acrylonitrile in this assay system. P450 1B1 is expressed constitutively in extrahepatic organs, including fetal tissue samples, and is highly inducible in various organs by 2,3,7,8-tetrachlorodibenzo-p-dioxin and related compounds in experimental animal models. Thus, activation of procarcinogens by P450 lB1 may contribute to human tumors of extrahepatic origin.
Adult↗
Cortical glucose metabolism in psychiatric wandering patients with vascular dementia.
The cerebral metabolic rate for glucose (CMRglc) in 18 patients with vascular dementia (VD) and 15 age-matched normal subjects was measured with positron emission tomography (PET) and the 18F-fluorodeoxyglucose technique. Nine of the 18 VD patients manifested abnormal wandering behavior with some psychiatric symptoms: they showed relatively spared CMRglc, which was higher than that of nonwandering patients, especially in the bilateral frontal lobes, left parietal lobe, left temporo-parieto-occipital region, left occipital lobe, and cerebellum. The results suggest that psychiatric wandering behavior of patients with VD could be correlated with partial sparing of CMRglc in the regions listed above.
Aged↗
Direct determination of pantoprazole enantiomers in human serum by reversed-phase high-performance liquid chromatography using a cellulose-based chiral stationary phase and column-switching system as a sample cleanup procedure.
A direct stereoselective reversed-phase HPLC method is described for the determination of the enantiomers of a proton pump inhibitor, pantoprazole (PAN), in human serum. The enantiomers were separated with high resolution on a cellulose-based chiral stationary phase (Chiralcel OJ-R) following on-line solid phase sample cleanup with a column-switching device. A mixture of acetonitrile and 50 mM sodium perchlorate was used as the mobile phase at a flow rate of 0.5 mliter/min. Pantoprazole enantiomers were detected by monitoring the column effluent with UV light at a wavelength of 290 nm. The calibration curve for each enantiomer was linear from 0.1 to 5.0 micrograms/mliter. Under these conditions, the determination of pantoprazole enantiomers in human serum can be achieved with satisfactory selectivity, sensitivity, precision, and accuracy. The described procedure is very simple and rapid since labor-intensive sample preparation is not required. The method was applied to the analyses of the serum samples obtained from a volunteer who received an 80-mg oral dose of racemic PAN. The samples showed the (+)/(-) isomer ratios ranging from 0.74 to 1.03 up to 6 h after dosing, indicating that there is only a small difference in the concentrations of (+)- and (-)-PAN.
2-Pyridinylmethylsulfinylbenzimidazoles↗
High rates of substrate hydroxylation by human cytochrome P450 3A4 in reconstituted membranous vesicles: influence of membrane charge.
CYP3A4 represents the most important form of human cytochrome P450 active in drug metabolism. Reconstitution of this enzyme has in the past been a major problem. Using purified cDNA-expressed CYP3A4 incorporated into membranous vesicles made from microsomal phospholipids, rates of nifedipine and testosterone oxidation of about 60 nmol/nmol P450/min were achieved, whereas similar reconstitution into dilauroyl-phosphatidylcholine micelles was unsuccessful. A higher Vmax for nifedipine oxidation was obtained in negatively charged vesicles as compared to neutral membranes, whereas the membrane charge did not influence the Km. It is concluded that the native function of CYP3A4 requires a negatively charged microsomal membrane.
Animals↗
Recombinant human cytochrome P450 1A2 and an N-terminal-truncated form: construction, purification, aggregation properties, and interactions with flavodoxin, ferredoxin, and NADPH-cytochrome P450 reductase.
Previous work from this laboratory indicated that the N-terminus of recombinant human cytochrome P450 (P450) 1A2 expressed in Escherichia coli is blocked (P. Sandhu, Z. Guo, T. Baba, M. V. Martin, R. H. Tukey, and F. P. Guengerich, (1994) Arch. Biochem. Biophys. 30, 168 -177). A modification of this construct was done to insert an extra 12 residues containing a thrombin-sensitive site just beyond the most N-terminal hydrophobic segment, and the protein was expressed, purified, and cut with thrombin. Treatment of E. coli membranes in which the P450 1A2 with 12 extra residues was present with thrombin did not release the truncated form, suggesting that the added thrombin site may be imbedded in the membrane. The N-terminal of the recombinant proteins were blocked but mild acid hydrolysis generated the expected Met residues as analyzed by Edman degradation. Laser light scattering studies indicated that purified thrombin-cleaved P450 1A2 (devoid of the usual N-terminal 25 residues or the first 36 residues of the wild-type protein) was still aggregated in the absence of detergent and that some nondenaturing detergents could reduce the apparent size to that of a tetramer. The N-terminal truncated protein was as catalytically active as full-length P450 1A2 but required a higher concentration of NADPH-P450 reductase. P450 1A2 exhibited catalytic activity in E. coli cells, and activity of the purified enzyme could be supported by E. coli flavodoxin and NADPH-flavodoxin reductase. Spinach ferredoxin and NADPH-ferredoxin reductase could also substitute for NADPH-P450 reductase. These artificial electron donors did not require phospholipid for oxidation reactions; however, phospholipid was required for optimal activity when either P450 1A2 or the truncated form was used with NADPH-P450 reductase. Rates of oxidation of 7-ethoxyresorufin were considerably higher for both P450 1A2 and the truncated form when NADPH-P450 reductase was replaced with the "oxygen surrogate" iodosylbenzene, indicating that P450 reduction and oxygen activation are normally limiting in this P450 1A2 reaction.
Amino Acid Sequence↗
7-Ethoxycoumarin O-deethylation catalyzed by cytochromes P450 1A2 and 2E1 in human liver microsomes.
7-Ethoxycoumarin O-deethylation has been used widely as a marker activity for assessing substrate specificities of cytochromes P450 (P450) in liver microsomes of mammals, and extensive studies have shown that in rats and mice the major catalysts are P450 1A1, 1A2, and 2B enzymes. In contrast to findings in experimental animal models, P450 2E1 has been reported to be a principal enzyme involved in 7-ethoxy-coumarin O-deethylation in human livers. In this study, we further examined the roles of individual forms of human P450 involved in 7-ethoxycoumarin O-deethylation using microsomes from different human liver samples and from human lymphoblastoid cells expressing human P450 enzymes and purified P450 enzymes isolated from the membrane of Escherichia coli expressing modified P450 proteins. Kinetic analysis showed that there were at least two different enzymes involved in 7-ethoxycoumarin O-deethylation in different human samples. Samples that contained high amounts of P450 2E1 in liver microsomes showed biphasic curves for O-deethylation with relatively high turnover numbers, whereas P450 1A2-rich samples tended to have low Km values with low Vmax values. Anti-human P450 2E1 antibodies inhibited markedly (P < 0.05) the 7-ethoxycoumarin O-deethylation activities catalyzed by human liver microsomes particularly when examined at a high substrate concentration (200 microM). However, we also found that anti-P450 1A2 antibodies suppressed O-deethylation activities only at a low substrate concentration (10 microM). Recombinant human P450 1A2 was found to have a low Km value for 7-ethoxycoumarin O-deethylation, whereas P450 2E1 showed a high Km value. Of the P450 enzymes examined, P450 1A1 gave the highest O-deethylation activities with a low Km value, although this enzyme is reported to be expressed extrahepatically in humans. Other human P450 enzymes, including P450 2A6, 2C10, 2D6, 3A4, and 3A5, did not show significant O-deethylation activities except that P450 2B6, a minor P450 component in human livers, was found to have a Vmax value similar to that of P450 1A2 and a Km value similar to that of P450 2E1. These results suggest that P450 1A2 is a low Km enzyme for 7-ethoxycoumarin O-deethylation in human liver microsome, although it has a low Vmax value than P450 2E1.
7-Alkoxycoumarin O-Dealkylase↗
Roles of cytochrome b5 in the oxidation of testosterone and nifedipine by recombinant cytochrome P450 3A4 and by human liver microsomes.
NADH-dependent testosterone 6 beta-hydroxylation and nifedipine oxidation activities could be reconstituted in systems containing cytochrome b5 (b5), NADH-b5 reductase, and bacterial recombinant cytochrome P450 (P450) 3A4 with a synthetic phospholipid mixture, cholate, MgCl2, and reduced glutathione. Replacement of NADH-b5 reductase with NADPH-P450 reductase produced an eightfold increase in testosterone 6 beta-hydroxylation activity. Further stimulation could be obtained when NADPH was used as an electron donor instead of NADH. Removal of b5 from the NADH- and NADPH-supported systems caused a 90% loss of testosterone 6 beta-hydroxylation activities in the presence of NADPH-P450 reductase but resulted in complete loss of the activities in the absence of NADPH-P450 reductase. These results suggested that about 10% of the activities was due to electron flow from NADPH-P450 reductase to P450 3A4 in the absence of b5. In the presence of testosterone and MgCl2, P450 3A4 was reduced by b5 and NADH-b5 reductase, although the rate of P450 3A4 reduction was much slower than that by NADPH-P450 reductase. Anti-human b5 immunoglobulin G (IgG) (purified using rabbit b5 affinity chromatography) inhibited testosterone 6 beta-hydroxylation activity catalyzed by human liver microsomes more strongly in NADH- than in NADPH-supported reactions. However, anti-rat NADPH-P450 reductase IgG inhibited microsomal activities in both NADH- and NADPH-supported systems to similar extents. Addition of NADH enhanced NADPH-supported testosterone and nifedipine oxidations in human liver microsomes. MgCl2 stimulated rates of reduction of b5 by NADPH-P450 reductase, but not by NADH-b5 reductase, in reconstituted systems. These results suggest that b5 is an essential component in P450 3A4-catalyzed testosterone hydroxylation and nifedipine oxidation in human liver microsomes. Our previous observation that rates of reduction of ferric P450 3A4 by NADPH-P450 reductase are accelerated by complexation with substrates and b5 is supported in this study.
Animals↗