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1.
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
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.
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
4.
The reduction-oxidation reactions of NADH and cytochrome to incipient oxygen insufficiency caused by nitrogen ventilation or hemorrhagic hypotension were examined in the exposed cerebral cortex of the cat. A comparison of the onset of redox changes with each procedure shows that cytochrome reduction precedes the reduction of mitochondrial NAD. This constitutes evidence that, in the living brain, NADH maintains its resting oxidation state at lower cellular oxygen tensions than cytochrome does, consistent with the differences in oxygen affinity these respiratory chain components exhibit during oxygen titration . 相似文献
5.
Leonard M. Hjelmeland Lewis Aronow James R. Trudell 《Biochemical and biophysical research communications》1977,76(2):541-549
Isotope effects for hydroxylation reactions catalyzed by cytochrome P-450 have usually been measured by comparing the overall reaction velocities of deuterated and nondeuterated substrates. Since the rate-limiting step is probably not the single reaction involving covalent bond cleavage, such an approach does not yield information about the primary isotope effect. We measured the primary kinetic isotope effect for benzylic hydroxylation by a method utilizing intramolecular competition, using the symmetrical substrate 1,3-diphenylpropane-1,1-d2. These experiments yield a value of , a larger effect than has previously been reported for benzylic hydroxylations. 相似文献
6.
P Debey E J Land R Santus A J Swallow 《Biochemical and biophysical research communications》1979,86(4):953-960
Several rate constants for one-electron reduction of cytochrome P450 are more rapid in the absence than in the presence of the specific substrate. The respective values for methyl viologen, nicotinamide adenine dinucleotide and the 1-methyl-4-(and -3-)carbamidopyridinium radicals are 2.6, 3.4, 6 and 35 × 107 M?1 s?1 without camphor, and 0.15, 0.1, 1.8 and 110 × 107 M?1 s?1 for the camphor complex. Hydrated electrons react with cytochrome P450 with a rate constant of 3.0 × 1010 M?1 s?1 whether camphor is bound or not, but little of the reduction takes place at the haem iron. No reduction of the haem iron by or could be detected, whether camphor is bound or not. 相似文献
7.
Bacterial bioluminescence-identification of fatty acid as product, its quantum yield and a suggested mechanism 总被引:5,自引:0,他引:5
By using radioactive decanal the direct transformation of this aldehyde to decanoic acid, with a quantum yield of 0.13, has been demonstrated. A mechanism analogous to that of other better understood bioluminescent reactions is proposed, leading to a product, as yet unisolated from the enzymic reaction, whose fluorescence spectrum is an excellent match for that of the luminescence.The extensive examination1,2,3 of the isolated bacterial luminescence system has resulted in the accepted outline shown. We wish to modify it, in accordance with the previous evidence, by suggesting that ’intermediates I and II‘ in Hastings' terminology2 are the same enzyme bound FMNH2 moiety. A lively controversy has surrounded the attempts to determine whether aldehyde exerts a purely catalytic role2 or is transformed in the reaction.4 If the aldehyde reacts, then the simplest product is the corresponding carboxylic acid, perhaps formed via the peracid. The most likely alternative reaction would involve enolistation and oxidation at the α-methylene group. We examined the second alternative fairly carefully, and found no evidence for it. We do not wish to report these results in detail at present, since we have now established that the acid corresponding to that formed in a normal autoxidation of the aldehyde is the product. Some indication of the nature of the products of the reaction is available.5Since the amount of product in the reaction is restricted to a very low level by the concentrations required, we labelled decanal with tritium at C-2 and thus were able to record the yield with some precision. Although recent work6 strongly implies that acid is formed stoichiometrically, the direct measurement of the quantum yield with respect to acid formation is necessary before a mechanism can be written. We have suggested a mechanism compatible with observations in this system, analogous to all cases of bioluminescence for which a mechanism is reasonably well established. This mechanism also leads to a product excited state with excellent agreement around pH7 in fluorescence wavelength to that of the luminescence. 相似文献
8.
Purified cytochrome from bovine adrenocortical mitochondria was incorporated into liposomes by the cholate-dilution method utilizing either dialysis or Sephadex gel filtration. Among synthetic phospholipids tested, dioleoylglycerophosphocholine showed the best stability during the incorporation of into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the showed two major populations on the elution pattern of the Sepharose 4B gel filtration, and were seen at a diameter of 200–600 Å and its aggregated forms. When the cytochrome was incorporated into dioleoylglycerophosphocholine liposomes or cholesterol-free adrenocortical mitochondrial liposomes, was less stable than in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal . Liposomal required cholesterol for its optimum reduction with adrenodoxin, adrenodoxin reductase, and NADPH in the presence of CO. About 70% of the total heme in the dioleoylglycerophosphocholine liposomes was reduced by the enzymatic reduction in the presence of cholesterol, indicating that 70% of the total molecules are exposed to the surface of the outer monolayer. In order to see the location of the heme in membrane, the dioleoylglycerophosphocholine-liposomal was subjected to treatment. This reagent destroyed the liposomal . These results suggest that the heme is located in the proximity of the reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane. 相似文献
9.
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
10.
Shakunthala Narasimhulu 《生物化学与生物物理学报:生物膜》1979,556(3):457-468
The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant () and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome is considerably restrained from expressing its maximum substrate binding potential at physiological temperature. In addition, the results indicate that the majority of the lipids apparently do not play a significant role in imposing constraint on the substratecytochrome binding reaction and in the temperature dependency of the . 相似文献
11.
Cytochrome b5 as electron donor to rabbit liver cytochrome P-450LM2 in reconstituted phospholipid vesicles 总被引:3,自引:0,他引:3
M Ingelman-Sundberg I Johansson 《Biochemical and biophysical research communications》1980,97(2):582-586
When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450LM2, cytochrome b5 enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or -nitroanisole about 5-fold. Cytochrome b5 did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b5-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H2O2 or in the system, thus strongly indicating cytochrome b5 being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase. 相似文献
12.
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
13.
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
14.
David F. Wilson Charles Owen Leena Mela Louis Weiner 《Biochemical and biophysical research communications》1973,53(1):326-333
The rate of respiration of suspensions of mitochondria in the presence of excess oxygen and substrate is shown to be dependent on the ratio of the concentration of adenosine triphosphate (ATP) to the product of the concentrations of adenosine diphosphate and orthophosphate. The mitochondrial respiratory chain is essentially in equilibrium with the reactions for ATP synthesis. The rate of mitochondrial respiration is controlled by the free energy requirement for ATP synthesis and this control is expressed on the rates of the reactions for reduction of the dehydrogenases by substrate and the oxidation of cytochrome by molecular oxygen. 相似文献
15.
The concept of high- and low-affinity reactions in bovine cytochrome c oxidase steady-state kinetics
(1) Analysis of the data from steady-state kinetic studies shows that two reactions between cytochrome c and cytochrome c oxidase sufficed to describe the concave Eadie-Hofstee plots ( and ). It is not necessary to postulate a third reaction of . (2) Change of temperature, type of detergent and type of cytochrome c affected both reactions to the same extent. The presence of only a single catalytic cytochrome c interaction site on the oxidase could explain the kinetic data. (3) Our experiments support the notion that, at least under our conditions (pH 7.8, low-ionic strength), the dissociation of ferricytochrome c from cytochrome c oxidase is the rate-limiting step in the steady-state kinetics. (4) A series of models, proposed to describe the observed steady-state kinetics, is discussed. 相似文献
16.
G W Ruddock J A Raleigh C L Greenstock 《Biochemical and biophysical research communications》1981,102(1):554-560
A steady-state competition system has been developed to investigate the reactions of the superoxide radical anion () with various peroxides, including the so-called Haber-Weiss reaction. Potassium superoxide dissolved in an oxygen-free solution of DMSO containing 18-dicyclohexyl-6-crown, is the source of . High pressure liquid chromatography is used as an assay system for reactivity, to detect and quantitate the yield of anthracene, formed as a major product in the reaction between and 9,10-dihydroanthrancene. Decrease in anthracene yields, in the presence of peroxide, may be used to indicate a possible competing reaction between and added peroxide. Complications involving peroxide-stimulated formation of anthraquinone derivatives are discussed. No evidence for a competing reaction between and peroxide can be detected up to a 10-fold excess of peroxide over 9,10-dihydroanthracene. 相似文献
17.
George Barany 《Analytical biochemistry》1980,109(1):114-122
An explicit set of general methods for the experimental determination of the rates k1 and k2 of consecutive pseudo-first-order reactions is described and discussed. These rely on the direct simultaneous analytical quantitation of the starting material, intermediate, and product of the reaction, and thus differ from present techniques based on measurement of coreactant consumption or coproduct appearance. The quantity is shown to define a good “envelope” approximation to product formation according to the simple law 100% [1 ? exp(?kenvt)]. The theory of envelopes is useful for comparing overall rates of reactions with widely differing values of . The kinetic pattern of thiolysis of dithiasuccinoyl amino acids to carbamoyl disulfide intermediates to product free amino acids is analyzed and shown to agree quantitatively with theory. 相似文献
18.
Cytochrome was extracted and purified from beef liver by a detergent method (cytochrome ). The hydrophilic moiety which carries the heme group (cytochrome ) was prepared by trypsin action upon pure cytochrome .Single-shelled lecithin liposomes form complexes with cytochromes up to a molar ratio of one protein for 35 phospholipids. The lipid-protein complexes were isolated by gel filtration on Sepharose 4B. They are hollow vesicles in which [3H]-glucose can be trapped. Their diameter is greater than that of the initial liposomes.Cytochrome does not interact with the vesicles. These results show that the hydrophobic tail is necessary for the binding and that the hydrophilic part of the protein is located on the outer face of the vesicles. This asymmetry is also proved by the action of reducing agents.Experiments with saturated phosphatidylcholines show that the protein interacts with the lipids both below the transition temperature . i.e. when the aliphatic chains are in a crystalline state, and above , when the alipathic chain are in a fluid state.1H NMR spectra show that even at the maximum cytochrome concentration the presence of the proteins does not markedly change the dynamics to the phospholipid molecules. An asymmetric single-shelled vesicle structure is proposed for the complex. 相似文献
19.
2-Hydroxymethyl-4-nitrophenyl trimethylacetate is rapidly converted, by an intramolecular pathway, to its benzyl ester counterpart in aqueous solutions of dilute buffers. Intramolecular acyl migration is favored by a factor of 105 over intermolecular transfer of the trimethylacetyl group to surrounding water molecules. The activation parameters of the reaction demonstrate that the rate acceleration is primarily entropic in origin. At constant pH, the apparent first-order rate constant for intramolecular acyl migration displays a linear dependence on the concentration of the basic component of the buffer. For catalysis by imidazole, a solvent deuterium isotope effect of is observed, in accord with a general base-catalyzed pathway. Similarities between intramolecular and intracomplex transacylations are discussed with the conclusion that the migration of a trimethylacetyl group from the phenolic oxygen atom of a 2-hydroxymethyl-4-nitrophenol to the adjacent benzylic oxygen atom provides an accurate model for acylation of the serine hydroxyl group at the active site of α-chymotrypsin by nitrophenyl esters. 相似文献
20.
The dependence of the mitochondrial respiratory rate on the reduction of cytochrome c has been measured as a function of the exogenous ratio and pH. The respiratory rate at values of less than 10-1m-1 is proportional to the reduction of cytochrome c and independent of pH from pH 6.5 to pH 8.O. The maximal turnover number (at 100% reduction) for cytochrome c is approximately 70 s?1. As the ratio is increased from 10?1m?1 to 104m?1, the respiration at any given level of reduction of cytochrome c is progressively inhibited. Greater inhibition is observed at more oxidized levels of cytochorme c with respiratory control values for oxidation of reduced cytochrome c exceeding 10. The behavior of mitochondrial respiratory control is shown to be quantitatively consistent with a proposed mechanism in which the regulation occurs in the reaction of oxygen with cytochrome oxidase. A steady-state rate expression is derived which fits the mitochondrial respiratory rate dependence on (i) the extramitochondrial ratio; (ii) the level of reduction of cytochrome c (or the intramitochondrial at different values; (iii) the pH of the suspending medium. This rate expression appears to correctly predict the relationships of the cytoplasmic ratio, the mitochondrial ratio, and the mitochondrial respiratory rate in intact cells as well as suspensions of isolated mitochondria. 相似文献