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1.
The effect of extra bound cytochrome b-5 on cytochrome P-450-dependent enzyme activities in liver microsomes 总被引:1,自引:0,他引:1
Binding of increasing amounts of detergent-purified cytochrome to rabbit liver microsomes produces a progressive inhibition of NADPH-cytochrome P-450 reductase activity which is accompanied by a similar inhibition of NADPH-supported benzphetamine demethylation. In contrast, NADH-cytochrome P-450 reductase activity in the enriched microsomes is markedly enhanced and this stimulation is accompanied by a similar increase in NADH-peroxidase activity, suggesting that cytochrome in these two reactions functions as an intermediate electron carrier to cytochrome P-450. 相似文献
2.
F. Peter Guengerich David P. Ballou Minor J. Coon 《Biochemical and biophysical research communications》1976,70(3):951-956
Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450LMredComplex I→Complex II→P-450LMox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome is added. The latter protein does not appear to function as an electron carrier in this process. 相似文献
3.
A gel-electrophoretically homogeneous preparation of cytochrome P-450 from liver microsomes of phenobarbital-pretreated rabbits 总被引:9,自引:0,他引:9
Cytochrome P-450 was purified from liver microsomes of phenobarbital-pretreated rabbits to a specific content of 16 to 17 nmoles per mg of protein with a yield of about 10 %. The purified cytochrome yielded only a single protein band on sodium dodecylsulfate-urea-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 45,000 was estimated for the protein. The preparation was free of cytochrome , NADH-cytochrome reductase, and NADPH-cytochrome reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome reductase preparation (purified by a detergent method) and phosphatidyl choline. 相似文献
4.
K Sugioka H Nakano T Noguchi J Tsuchiya M Nakano 《Biochemical and biophysical research communications》1981,100(3):1251-1258
The system, which contains NADPH, purified cytochrome P-450 reductase, and adriamycin, produces H2O2 and in appreciable amounts with oxygen consumption and NADPH oxidation under aerobic conditions. Such an adriamycin-induced NADPH oxidation system, however, does not cause the decomposition of unsaturated fatty acids in microsomal phospholipid micelles, suggesting no direct participation of the active oxygen species and semiquinone radicals of adriamycin in lipid peroxidation. Adriamycin produces a co-ordination complex with Fe3+ and ADP, which, but no Fe3+-ADP complex, could be reduced by NADPH-cytochrome P-450 reductase at the expence of NADPH. The decomposition of unsaturated fatty acids in phospholipid micelles is achieved by the Fe3+-ADP-adriamycin complex and strikingly enhanced by enzymatically reduced iron-ADP-adriamycin complex. 相似文献
5.
Highly purified divalent and monovalent antibodies against cytochrome , anti- immunoglobulin G (IG) and anti- Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti- IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti- Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline. 相似文献
6.
NADPH reduces both liver microsomal cytochrome P-450 and cytochrome . In the presence of CO, ferrous cytochrome P-450 can slowly transfer electrons to amaranth, an azo dye. This reaction is followed by the reoxidation of cytochrome which proceeds at essentially the same rate as does cytochrome P-450 oxidation. It is suggested that cytochrome directly reduces cytochrome P-450 in rat liver microsomes. 相似文献
7.
J L Purvis J A Canick S A Latif J H Rosenbaum J Hologgitas R H Menard 《Archives of biochemistry and biophysics》1973,159(1):39-49
The lifetime of different microsomal steroidogenic enzymes and the cytochrome components of the NADPH-cytochrome P-450 pathway have been determined in rat testis by measuring their decrease logarithmically after hypophysectomy. Although both cytochrome P-450 and 17α-hydroxylase show biphasic decay curves, the first decay curve contains 89–94% of the cytochrome P-450 and 17α-hydroxylase levels. Steroidogenic enzymes which are located mainly in the leydig cells, decay much faster than microsomal protein, days, which represents mainly decay of tubular protein. The similarity between the major half-life of cytochrome P-450, days, 17α-hydroxylase, days and the C17–C20 lyase, days and the uniformity of their response to human chorionic gonadotrophin (HCG) provides additional evidence that these two steroidogenic enzymes require cytochrome P-450. Both the 17α-hydroxylase and the C17–C20 lyase were shown to have a constant activity per nmole of cytochrome P-450 during a sixfold change in the level of cytochrome P-450 brought about by HCG treatment of rats with intact pituitaries. The decay of 17β-hydroxysteroid dehydrogenase, days, was slower than P-450 dependent enzymes. Rats with intact pituitaries are not under maximal stimulation by endogenous LH because addition of HCG increases the levels of microsomal and mitochondrial cytochrome P-450 220 and 1620%, respectively. The rates of synthesis during the increase from one cytochrome P-450 level to another was calculated at testes/day for microsomal cytochrome P-450 and 0.10 nmoles/2 testes/day for mitochondrial cytochrome P-450. Treatment of hypophysectomized rats with HCG results in large increases of cytochrome P-450, 17α-hydroxylase, C17–C20 lyase and 5α-reductase, but not cytochrome b5, microsomal protein, 7α-hydroxylase, or the 17β-hydroxysteroid dehydrogenase. While it is clear that the two cytochrome P-450 dependent hydroxylases involved in steroidogenesis and the 5α-reductase are under the control of gonadotrophin, it is not clear how 17β-hydroxysteroid dehydrogenase levels are maintained or in what manner the 5α-reductase level is controlled in mature animals. 相似文献
8.
Theodore G. Gabig Bruce A. Lefker 《Biochemical and biophysical research communications》1984,118(2):430-436
The resolved flavoprotein and cytochrome b559 components of the NADPH dependent generating oxidase from human neutrophils were the subject of further study. The resolved flavoprotein, depleted of cytochrome b559, was reduced by NADPH under anaerobic conditions and reoxidized by oxygen. NADPH dependent generation by the resolved flavoprotein fraction was not detectable, however it was competent in the transfer of electrons from NADPH to artificial electron acceptors. The resolved cytochrome b559, depleted of flavoprotein, demonstrated no measureable NADPH dependent generating activity and was not reduced by NADPH under anaerobic conditions. The dithionite reduced form of the resolved cytochrome b559 was rapidly oxidized by oxygen, as was the cytochrome b559 in the intact oxidase. 相似文献
9.
Mitsukazu Kitada Chiyo Yamazaki Kouzi Hirota Haruo Kitagawa 《Biochemical and biophysical research communications》1980,93(4):1020-1026
Cytochrome P-450 was purified from phenobarbital-treated guinea pigs to a specific content of 19.8 nmoles per mg of protein, and was free of cytochrome b5 and NADPH-cytochrome reductase. The purified cytochrome P-450 gave a single protein band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 49,000 was estimated. Benzphetamine N-demethylation activity could be reconstituted by mixing the purified cytochrome, NADPH-cytochrome reductase and phosphatidylcholine. 相似文献
10.
The role of cytochrome 5 in the -nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome 5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome 5 reductase, and Triton X-100 in addition to cytochrome 5. The omission of cytochrome 5 from the complete system entirely abolished the activity. These results clearly show that cytochrome 5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome 5 in such a way that the second electron is transferred from cytochrome 5 and thus exhibits the demethylase activity. 相似文献
11.
M.F. Budyka A.M. Khenkin A.A. Shteinman 《Biochemical and biophysical research communications》1981,101(2):615-622
Oxygenation of heme-mercaptide as well as spectroscopic characteristics of the dioxygen complex formed have been studied. Absorption and magnetic circular dichroism spectra of the O2 complex support the retention of mercaptide in the heme fifth position. A release of in the decomposition of the oxygenated complex and an independent formation of the latter from hemine-dimercaptide and together with electron paramagnetic resonance and Mössbauer data support the O2 presence in the heme coordination sphere. The similarity of optical and magnetic circular dichroism spectra and the closeness of the ratio for oxy-heme-mercaptide and oxycytochrome P450 unequivocally confirm the presence of an axial cystein mercaptide ligand in oxycytochrome P450. 相似文献
12.
Bengt Jernström Jorge Capdevila Sten Jakobsson Sten Orrenius 《Biochemical and biophysical research communications》1975,64(3):814-822
Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino--octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome 5 and NADPH-cytochrome reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found. 相似文献
13.
H A Sasame J R Gillette M R Boyd 《Biochemical and biophysical research communications》1978,84(2):389-395
An antibody prepared against purified rat liver NADPH-cytochrome reductase inhibited both the pulmonary and hepatic microsomal covalent binding of 4-ipomeanol as well as the respective NADPH-cytochrome reductase activities, findings which are consistent with previous studies which indicated the participation of cytochrome P450 in the metabolic activation of the toxin. An antibody prepared against purified rat liver cytochrome b5, which strongly inhibited both the rat hepatic and pulmonary NADH-dependent cytochrome reductases, and was inactive against the respective NADPH-dependent cytochrome reductases, had little effect on metabolic activation of 4-ipomeanol by hepatic microsomes, but strongly inhibited both the NADH-supported and the NADPH-supported pulmonary microsomal metabolism and covalent binding of the compound. These results suggest that metabolic activation of 4-ipomeanol involves a two-electron transfer in which transfer of the second electron via cytochrome b5 is rate-limiting in lung microsomes. 相似文献
14.
Yoshiyuki Ichikawa 《生物化学与生物物理学报:生物膜》1975,394(3):406-415
Optical and magnetic studies were made on subfractions of rabbit kidney cortex. Cytochrome and cytochrome mixed function oxidase systems were localized mainly in the brush border membranes and microsomes. Cytochrome mixed function oxidases in the membranes comprised both an NADPH-dependent system and an NADH-dependent system. 相似文献
15.
M. Tegoni J.M. Janot M.C. Silvestrini M. Brunori F. Labeyrie 《Biochemical and biophysical research communications》1984,118(3):753-759
Spectral redox titrations of flavin and cytochrome b2 moieties of flavocytochrome b2 were achieved in the absence and in the presence of pyruvate under equilibrium conditions at 18° C; direct measurements of spin flavosemiquinone proportions have been carried out by EPR determinations at the same temperature. Our results show that the equilibria involving flavin are largely affected by the presence of pyruvate; the semiquinone proportion markedly increases almost till unit near half-reduction of cytochrome b2; at 10 mM pyruvate, the dismutation constant, increases by a factor ≥ 10. 相似文献
16.
José Remacle 《生物化学与生物物理学报:生物膜》1980,597(3):564-576
The in vitro incorporation of cytochrome into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome than did microsomal preparations; 60% of this cytochrome could not be reduced by the NADH-cytochrome reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome . These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell. 相似文献
17.
K M Ivanetich I Aronson I D Katz 《Biochemical and biophysical research communications》1977,74(4):1411-1418
In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome 5 or NADPH-cytochrome reductase . 相似文献
18.
(1) +/electron acceptor ratios have been determined with the oxidant pulse method for cells of denitrifying Paracoccus denitrificans oxidizing endogenous substrates during reduction of O2, NO?2 or N2O. Under optimal H+-translocation conditions, the ratios , , for reduction to N2 and for reduction to N2O were 6.0–6.3, 4.02, 5.79 and 3.37, respectively. (2) With ascorbate/N,N,N′,N′-tetramethyl-p-phenylenediamine as exogenous substrate, addition of NO?2 or N2O to an anaerobic cell suspension resulted in rapid alkalinization of the outer bulk medium. , for reduction to N2 and for reduction to N2O were ?0.84, ?2.33 and ?1.90, respectively. (3) The ratios, mentioned in item 2, were not altered in the presence of and the triphenylmethylphosphonium cation. (4) A simplified scheme of electron transport to O2, NO?2 and N2O is presented which shows a periplasmic orientation of the nitrite reductase as well as the nitrous oxide reductase. Electrons destined for NO?2, N2O or O2 pass two H+-translocating sites. The acceptor ratios predicted by this scheme are in good agreement with the experimental values. 相似文献
19.
Peter Nicholls 《BBA》1976,430(1):13-29
1. Formate inhibits cytochrome oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome . The inhibition increases with decreasing pH, indicating that HCOOH may be the inhibitory species.2. Formate induces a blue shift in the absorption spectrum of oxidized cytochrome ) and in the half-reduced species (). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of minus , free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome ) and azide (which induces peak shifts of cytochrome towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome is faster than its rate of dissociation from , especially in the presence of cytochrome . The for formate inhibition of respiration is a function of the reduction state of the system, varying from 30 mM (100% reduction) to 1 mM (100% oxidation) at pH 7.4, 30 °C.4. Succinate-cytochrome reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]2.5. Formate inhibition of ascorbate plus oxidation by intact rat liver mitochondria is partially released by uncoupler addition. Formate is permeable through the inner mitochondrial membrane and no differences in ‘on’ or ‘off’ inhibition rates were observed when intact mitochondria were compared with submitochondrial particles.6. NADH-cytochrome reductase activity is unaffected by formate in submitochondrial particles, but mitochondrial oxidation of glutamate plus malate is subject both to terminal inhibition at the cytochrome level and to a slow extra inhibition by formate following uncoupler addition, indicating a third site of formate action in the intact mitochondrion. 相似文献
20.
(1) Aqueous solutions of 1–10 μM ferricytochrome c treated with 100 μM–100 mM H2O2 at pH 8.0 emit chemiluminescence with quantum yield and absolute maximum intensity per cm3 (λ = 440), and exhibit exponential decay with a rate constant of 0.15 s?1. (2) The emission spectrum of the chemiluminescence covers the range 380–620 nm with the maximum at 460 ± 10 nm. (3) Neither cytochrome c nor haemin fluoresce in the spectral region of the chemiluminescence. In the reaction course with H2O2, a weak fluorescence in the region 400–620 nm with λmax = 465–510 nm (λexc 315–430 nm) gradually arises. This originates from tryptophan oxidation products of the formylkynurenine type or from imidazole derivatives, respectively. (4) Frozen solutions (77 K) of cytochrome c exhibit phosphorescence typical of tryptophan (λexc = 280 nm, λem = 450 nm). During the peroxidation, an additional phosphorescence gradually appears in the range 480–620 nm with λmax = 530 nm (λexc = 340 nm). This originates from oxidative degradation products of tryptophan. (5) There are no red bands in the chemiluminescence spectra of cytochrome c or haemin. This result suggests that singlet molecular oxygen is not involved in either peroxidation or chemiluminescence. (6) The haem Fe3+ group and H2O2 appear to be crucial for the chemiluminescence. It is suggested that the generation of electronically excited, light-emitting states is coupled to the production of conformational out-of-equilibrium states of peroxy-Fe-protoporphyrin IX compounds. 相似文献