共查询到20条相似文献,搜索用时 15 毫秒
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D E Williams R T Okita D R Buhler B S Masters 《Archives of biochemistry and biophysics》1984,231(2):503-510
Microsomes from liver or kidney of untreated rainbow trout hydroxylated lauric acid specifically at the (omega-1) position. Turnover numbers for liver (2.72 min-1) and kidney (14.1 min-1) were decreased seven- and twofold, respectively, following treatment with beta-naphthoflavone. Laurate hydroxylation activity from untreated trout hepatic microsomes was sensitive to inhibition by SKF-525A, but was not sensitive to metyrapone and only partially inhibited by alpha-naphthoflavone. The temperature optimum of laurate (omega-1) hydroxylation in trout liver microsomes was 25-30 degrees C. The Km and Vmax for (omega-1)- hydroxylaurate formation was 50 microM and 1.63 nmol min-1 mg-1, respectively, in liver and 20 microM and 3.95 nmol min-1 mg-1, respectively, in kidney from untreated trout microsomes. (omega-1) Hydroxylation of laurate, in both liver and kidney microsomes, was sensitive to an antibody raised against a previously purified cytochrome P-450 isozyme (LM2) of trout liver microsomes, which has been shown to be active towards aflatoxin B1. Antibody to the major isozyme of cytochrome P-450 ( LM4b , active towards benzo(a)pyrene) induced by beta-naphthoflavone did not inhibit (omega-1) hydroxylation of laurate in microsomes from untreated or beta-naphthoflavone-treated trout. 相似文献
3.
Hans Grasdalen L.E. Göran Eriksson Anders Ehrenberg Dan Bäckström 《Biochimica et Biophysica Acta (BBA)/General Subjects》1978,541(4):521-527
Cytochrome P-450 in microsomes from liver of phenobarbital treated and control rats has been studied by light absorption and by magnetic resonance methods (EPR and NMR). The nuclear relaxation rate of water protons was measured for microsomal suspensions in the presence of various reactants of Type I and II. The change of relaxation rates correlates well with the spin state conversion of the heme iron. No competition between eventual inner-sphere water molecules and the reactants seems to occur. The temperature dependence of the low spin to high spin equilibrium was studied by light absorption and was accounted for in the temperature variation of the molar relaxation rates of the two spin states. 相似文献
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5.
A reconstituted mixed-function oxidase system containing cytochrome P-450, cytochrome P-450 reductase, phosphatidylcholine, and NADPH catalyzed the reduction of 13-hydroperoxy-9,11-octadecadienoic acid to 13-hydroxy-9,ll-octadecadienoic acid. Activity was stimulated by the addition of type I substrates, while carbon monoxide and oxygen inhibited the reaction. Perfluoro-n-hexane stimulated the reduction of lipid hydroperoxide to lipid alcohol in the reconstituted system but not by cytochrome P-450 alone. Incubation of cytochrome P-450 with only lipid hydroperoxide resulted in destruction of the hemoprotein. Addition of substrates such as aminopyrine decreased cytochrome P-450 destruction. Addition of reducing equivalents from a reconstituted electron transport system also decreased cytochrome P-450 destruction. 相似文献
6.
K Totani T Iizuka H Shimada R Makino Y Ishimura 《Archives of biochemistry and biophysics》1983,222(1):207-215
Effects of pH on the ligand-binding reactions of ferric heme in cytochrome P-450 from Pseudomonas putida (camphor 5-monooxygenase, EC 1.14.15.1) were studied by using cyanide, N-methylimidazole, pyridine, and ethylisocyanide as ligands. In all cases, affinity of the ferric heme for the ligand was found to increase as pH of the medium was raised from around 6 to 9. Depending on the ligand, the increase was 10- to 1000-fold and the shapes of their pH-affinity curves were remarkably different. Analyses such pH profiles disclosed the presence of a dissociable group in the enzyme with a pK value of approximately 9.5 and that its ionization greatly enhanced the affinity of the heme for ligands. When a dissociable ligand such as hydrogen cyanide and N-methylimidazole was used, the dissociated form of the ligand had a higher affinity toward the heme than the undissociated form. The shapes of the pH-affinity curves were successfully simulated as overlapping curves of ionization reactions of the ligand and the dissociable group. In addition, size of the ligand molecule was shown to be also important in the binding reaction: relatively large molecules such as pyridine, ethylisocyanide, and N-methylimidazole bound to the enzyme in a competitive manner against d-camphor concentration, whereas the binding of a smaller molecule such as cyanide was inhibited by the substrate in a noncompetitive manner. On the basis of these findings, control mechanisms for the ligand-binding reactions of the cytochrome P-450 from P. putida are discussed. 相似文献
7.
Differences in the mechanism of NADPH- and cumene hydroperoxide-supported reactions of cytochrome P-450 总被引:2,自引:0,他引:2
A study has been carried out on the association of aldolase with the human erythrocyte membrane. It has been shown that the conditions employed during hypotonic hemolysis affect the amount of aldolase that remains bound to the cell membrane. Thus, the in vivo nature of this binding cannot be ascertained by this technique. Therefore, a method has been developed in which aldolase is crosslinked with glutaraldehyde to the inner surface of the membrane in intact red blood cells. Under the specified conditions, over 90% of the intracellular aldolase can be crosslinked to the membrane with less than 10% of the hemoglobin becoming bound. These results suggest that the localization of aldolase in situ is on or near the inner surface of the membrane. The amount of aldolase bound to the membrane following crosslinking can be decreased by preincubating the cells with cytoskeletal agents such as cytochalasin B, colchicine, and vinblastine sulfate. The in vitro binding of aldolase to the purified spectrin-actin and F-actin complexes was studied. Aldolase bound both complexes very tightly (KD ? 10?9m) and this binding could be inhibited by cytochalasin B, but not by colchicine. A competition binding study was carried out to determine if the binding of aldolase to F-actin involved specific interactions. Neither bovine serum albumin nor cytochrome c significantly inhibited the binding of aldolase to F-actin when each was present at equimolar concentrations with aldolase. However, glyceraldehyde 3-phosphate dehydrogenase inhibited aldolase binding to F-actin and when present at equimolar concentrations with aldolase completely blocked the association. The association of aldolase and other glycolytic enzymes with the erythrocyte membrane is discussed and it is postulated that aldolase could be localized in vivo on the inner surface of the membrane by attachment to actin or a spectrin-actin complex. 相似文献
8.
Anaerobic dehalogenation of halothane by reconstituted liver microsomal cytochrome P-450 enzyme system 总被引:1,自引:0,他引:1
K Fujii N Miki T Sugiyama M Morio T Yamano Y Miyake 《Biochemical and biophysical research communications》1981,102(1):507-512
Cytochrome P-450 from liver microsomes of phenobarbital-treated rabbits catalyzed anaerobic dehalogenation of halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) when combined with NADPH and NADPH-cytochrome P-450 reductase. Cytochromes P-450B1 and P-448 from liver microsomes of untreated rabbits were less active. Triton X-100 accelerated the reaction. Unlike anaerobic dehalogenation of halothane in microsomes, the major product was 2-chloro-1,1,1-trifluoroethane and 2-chloro-1,1-difluoroethylene was negligible. These products were not detected under aerobic conditions, and dehalogenation activity was inhibited by carbon monoxide, phenyl isocyanide and metyrapone. 相似文献
9.
Paul R.Ortiz De Montellano Bruce A. Mico James M. Mathews Kent L. Kunze Gerald T. Miwa Anthony Y.H. Lu 《Archives of biochemistry and biophysics》1981,210(2):717-728
The autocatalytic destruction of cytochrome P-450 by the following six substrates has been investigated in vivo and in vitro with microsomal and purified, reconstituted rat liver enzymes: 2-isopropyl-4-pentenamide (AIA), 1-ethinylcyclopentanol, 17α-propadienyl-19-nortestosterone, fluroxene, 5,6-dichloro-1,2,3-benzothiadiazole (DCBT), and 1-aminobenzotriazole (ABT). Administration of the first three substrates to rats pretreated with either phenobarbital (Pb) or 3-methylcholanthrene (3-MC), or their incubation with hepatic microsomes from such rats, produced a larger decrease in cytochrome P-450 levels in the membranes from Pb- than 3-MC-treated rats. Comparable losses, however, were observed in microsomes from rats pretreated with both Pb and 3-MC when the last three agents were used. Similar experiments were carried out using the major cytochrome P-450 isozymes purified from liver microsomes of Pb- or 3-MC-treated rats. The Pb isozyme was inactivated during catalytic turnover of all six substrates while only three substrates (DCBT, ABT, and fluroxene) were found to inactivate the 3-MC isozyme. Oxygen consumption studies with purified enzymes have shown that AIA is not a measurable substrate for the 3-MC isozyme, a fact which explains its failure to inactivate this isozyme. Similar studies with the Pb isozyme establish that one enzyme molecule is inactivated for approximately every 230–320 AIA molecules processed by the enzyme. 相似文献
10.
Immunochemical evidence for a role of cytochrome P-450 in liver microsomal ethanol oxidation 总被引:5,自引:0,他引:5
Antibodies to cytochrome P-450 isozyme 3a, the ethanol-inducible isozyme in rabbit liver, were used to determine the role of this enzyme in the microsomal oxidation of alcohols and the p-hydroxylation of aniline. P-450 isozymes, 2, 3b, 3c, 4, and 6 did not crossreact with anti-3a IgG as judged by Ouchterlony double diffusion, and radioimmunoassays indicated a crossreactivity of less than 1%. Greater than 90% of the activity of purified form 3a toward aniline, ethanol, n-butanol, and n-pentanol was inhibited by the antibody in the reconstituted system. The catalytic activity of liver microsomes from control or ethanol-treated rabbits was unaffected by the addition of either desferrioxamine (up to 1.0 mM) or EDTA (0.1 mM), suggesting that reactions involving the production of hydroxyl radicals from H2O2 and any contaminating iron in the system did not make a significant contribution to the microsomal activity. The addition of anti-3a IgG to hepatic microsomes from ethanol-treated rabbits inhibited the metabolism of ethanol, n-butanol, n-pentanol, and aniline by about 75, 70, 80, and 60%, respectively, while the inhibition of the activity of microsomes from control animals was only about one-half as great. The rate of microsomal H2O2 formation was inhibited to a lesser extent than the formation of acetaldehyde, thus suggesting that the antibody was acting to prevent the direct oxidation of ethanol by form 3a. Under conditions where purified NADPH-cytochrome P-450 reductase-catalyzed substrate oxidations was minimal, the P-450 isozymes other than 3a had low but significant activity toward the four substrates examined. The residual activity at maximal concentrations of the antibody most likely represents the sum of the activities of P-450 isozymes other than 3a present in the microsomal preparations. The results thus indicate that the enhanced monooxygenase activity of liver microsomes from ethanol-treated animals represents catalysis by P-450 isozyme 3a. 相似文献
11.
H Sakurai S Shimomura K Ishizu 《Biochemical and biophysical research communications》1981,101(4):1102-1108
A new cytochrome P-450 model that simulates the unusual spectral and substrate-oxidation properties of cytochrome P-450 is proposed. The complex, consisting of glutathione(GSH), hemin and pyridine(py), exhibits similar optical and EPR spectra to cytochrome P-450 in ferric low-spin state. On omission of py, a ferric high-spin state was produced. On exposure of the GSH-hemin-py complex to CO, a characteristic absorption band appeared at 450nm, like that typical of cytochrome P-450. Two types of spectral changes were observed when aminopyrine or phenobarbital (Type I) and aniline or quinoline (Type II) were added to the GSH-hemin complex. Hydroxylation, dealkylation and aromatic methyl migration activities were observed with the GSH-hemin complex. 相似文献
12.
A.J. Marinello H.L. Gurtoo R.F. Struck B. Paul 《Biochemical and biophysical research communications》1978,83(4):1347-1353
Cyclophosphamide (CP) metabolites, acrolein and 4-hydroxy-CP, were found to denature rat liver microsomal cytochrome P-450, whereas another metabolite, phosphoramide mustard, CP or its analog Ifosfamide had no effect. The denaturation produced by CP metabolites could be blocked by cysteine, suggesting an interaction between CP metabolite(s) and sulfhydryl groups in cysteine and probably in cytochrome P-450. These studies might explain the biochemical basis of the specific depression of various microsomal mixed function oxygenase activities produced by high doses of CP. 相似文献
13.
Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay 总被引:2,自引:0,他引:2
The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form. 相似文献
14.
Laurence S. Kaminsky F.Peter Guengerich Ghazi A. Dannan Steven D. Aust 《Archives of biochemistry and biophysics》1983,225(1):398-404
R- and S-warfarin metabolite profiles (regio- and stereoselectivity) have been determined with hepatic microsomes from untreated rats and rats treated with nine individual polybrominated biphenyl (PBB) congeners, a commercial mixture of PBBs, and, for comparison with phenobarbital and 3-methylcholanthrene. The metabolic rates have been correlated with cytochrome P-450 (P-450) isozyme concentrations in the microsomes determined by immunochemical quantitation techniques (G. A. Dannan, F. P. Guengerich, L. S. Kaminsky, and S. D. Aust, (1983) J. Biol. Chem., 258, 1282–1288). The warfarin hydroxylase activities of the P-450 isozyme components of the various microsomal preparations (F. P. Guengerich, G. A. Dannan, S. T. Wright, M. V. Martin, and L. S. Kaminsky (1982) Biochemistry, 21, 6019–6030) were multiplied by the corresponding isozyme concentrations to obtain an assessment of the potential warfarin hydroxylase capacity of the microsomes, and the results were compared with actual activities. The results of these studies and comparisons indicate that substrate regio- and stereoselectivities of microsomal-bound P-450s are essentially retained on purification of the isozymes to homogeneity and reconstitution, that warfarin metabolism by microsomal preparations can be used to predict microsomal P-450 isozyme compositions, and that microsomal warfarin hydroxylase activity is greater than would be predicted based on the approx 20:1 ratio of P-450 to NADPH-P-450 reductase in the microsomes and on the known activities of constituent isozymes. Two P-450 isozymes which are induced by treatment of rats with phenobarbital appear to be more tightly linked to NADPH-P-450 reductase than does an isozyme induced by β-naphthoflavone. 相似文献
15.
Effect of cobalt chloride and 3-amino-1,2,4-triazole on the induction of cytochrome P-450 synthesis by phenobarbitone in rat liver 总被引:2,自引:0,他引:2
Administration of cobalt chloride and 3-amino-1,2,4-triazole leads to a suppression of phenobarbitone-mediated increase in total cytochrome P-450 as well as cytochrome P-450b contents of the liver. This suppression is due to a decrease in the content of the protein species which is the result of a decrease in its rate of synthesis as measured in vivo and in vitro. Cobalt chloride as well as 3-amino-1,2,4-triazole treatments lead to a decrease in the translatability of cytochrome P-450b RNA without affecting total protein synthesis. It is a possibility that a small pool of heme regulates the RNA levels for the cytochrome P-450 species. 相似文献
16.
Y Aoyama Y Yoshida S Kubota H Kumaoka A Furumichi 《Archives of biochemistry and biophysics》1978,185(2):362-369
NADPH-cytochrome c reductase of yeast microsomes was purified to apparent homogeneity by solubilization with sodium cholate, ammonium sulfate fractionation, and chromatography with hydroxylapatite and diethylaminoethyl cellulose. The purified preparation exhibited an apparent molecular weight of 83,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reductase contained one molecule each of flavin-adenine dinucleotide and riboflavin 5′-phosphate, though these were dissociative from the apoenzyme. The purified reductase showed a specific activity of 120 to 140 μmol/min/mg of protein for cytochrome c as the electron acceptor. The reductase could reduce yeast cytochrome P-450, though with a relatively slow rate. The reductase also reacted with rabbit liver cytochrome P-450 and supported the cytochrome P-450-dependent benzphetamine N-demethylation. It can, therefore, be concluded that the NADPH-cytochrome c reductase is assigned for the cytochrome P-450 reductase of yeast. The enzyme could also reduce the detergent-solubilized cytochrome b5 of yeast. So, this reductase must contribute to the electron transfer from NADPH to cytochrome b5 that observed in the yeast microsomes. 相似文献
17.
F J Fernández-Belda F García-Carmona F García-Cánovas J A Lozano J C Gómez-Fernández 《Archives of biochemistry and biophysics》1982,215(1):40-46
Purified F1-ATPase is slowly inactivated by interaction, in a preincubation medium, with its substrate MgATP. Interaction with Mg2+ before addition of ATP to the preincubation medium is essential to induce the inactivation. This inactivation is different from other Mg2+-induced inhibitions previously described. Free ATP concentration is implicated in the inactivation process and a linear relationship can be established between this concentration and the number of turnovers which are necessary for total inactivation. ITP, 2′-dATP, and GTP can also induce inactivation. Although ITP and GTP are hydrolyzed at a lower rate than ATP and 2′-dATP, they induce inactivation after a smaller number of turnovers than the latter. This process closely follows a kinetics of the type described for suicide enzymes. A reaction scheme is suggested and discussed. 相似文献
18.
David E. Williams Robert R. Becker David W. Potter F.Peter Guengerich Donald R. Buhler 《Archives of biochemistry and biophysics》1983,225(1):55-65
NADPH-cytochrome P-450 reductase has been purified to apparent homogeneity from liver microsomes of β-naphthoflavone-treated rats and rainbow trout. The apparent monomeric molecular weights were 75,000 and 77,000 for the rat and trout, respectively. Differences in amino acid composition were observed, particularly for lysine, glycine, threonine, and tyrosine. Analysis of the flavin composition showed that there were 0.97 mol of FAD and 0.92 mol of FMN per mol of rat reductase, whereas the values for the trout enzyme were 1.06 and 0.76 for FAD and FMN, respectively. Trout NADPH-cytochrome c reductase was inhibited by anti-rat antibody, but not to the same extent as was the rat enzyme. No precipitin lines between the trout reductase and rat antibody were observed on Ouchterlony plates. Peptide patterns, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, following limited proteolysis were also markedly different. The trout enzyme was as effective, catalytically, as the rat enzyme in a reconstituted system that contained purified rat cytochrome P-448 and lipid. Comparison of ethoxyresorufin-O-deethylase temperature profiles with various combinations of purified trout and rat P-448, reductase, and lipid, in membranous and nonmembranous reconstitution systems, demonstrated that the lower temperature optimum in trout microsomes could only be reproduced when all three trout components were incorporated into liposomes. These results suggest that it is the structural organization of the mixed-function oxidase enzymes and lipid within trout microsomes which were responsible for the lower temperature optimum compared to rat. 相似文献
19.
M Paye P Beaune P Kremers F P Guengerich F Letaw-Goujon J Gielen 《Biochemical and biophysical research communications》1984,122(1):137-142
An enzyme linked immunosorbent assay (ELISA) using monoclonal and polyclonal antibodies has been developed to quantify individual cytochrome P-450 isoenzymes in microsomal preparations, namely UT-A and PB-B. This very sensitive method can be used for the rapid processing of large quantities of determinations and requires only limited amounts of antibodies. 相似文献
20.
Y Yoshida Y Aoyama T Nishino H Katsuki U S Maitra V P Mohan D B Sprinson 《Biochemical and biophysical research communications》1985,127(2):623-628
An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity. 相似文献