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1.
The addition of cholate to the microsomes at 37.5°C resulted in a striking decrease in the apparent substrate dissociation constant (K′s) and its temperature dependency. The microsomal membranes depleted of 80% of the lipids preserved the temperature dependency of the Ks and exhibited breaks in the Van't Hoff plot at the characteristic temperature of the lipids phase transition. The results indicate that the cytochrome P-450 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 P-450 binding reaction and in the temperature dependency of the Ks.  相似文献   

2.
NADPH reduces both liver microsomal cytochrome P-450 and cytochrome b5. 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 b5 which proceeds at essentially the same rate as does cytochrome P-450 oxidation. It is suggested that cytochrome b5 directly reduces cytochrome P-450 in rat liver microsomes.  相似文献   

3.
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 p-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 O2? 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   

4.
Binding of increasing amounts of detergent-purified cytochrome b5 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 b5 in these two reactions functions as an intermediate electron carrier to cytochrome P-450.  相似文献   

5.
The association of fatty acids, androstane, phosphatidylcholine, phosphatidylethanolamine, and phosphatidic acid with purified and phospholipid-vesicle reconstituted cytochrome P-450 was studied by spin labeling. Spin-labeled fatty acids were found to be motionally restricted by cytochrome P-450 in both phospholipid vesicles and in microsomes to a much greater extent than spin-labeled phospholipids. The equilibrium of spin-labeled fatty acid between the bulk membrane lipid and the protein interface could be shifted towards an increased amount in the bulk phospholipid phase by the addition of oleic acid or lysophosphatidylcholine, but not by sodium cholate. Microsomes from different animals showed a variable extent of motional restriction of fatty acids, independent of pretreatment of the animals with phenobarbital or β-naphthoflavone, of cytochrome P-450 content, of the presence of type I and type II substrates for cytochrome P-450. These differences are attributed to the presence of varying amounts of lipid breakdown products in the microsomal membrane such as lysolipids or fatty acids which compete with the externally added spin-labeled fatty acids, or with spin-labeled androstane for the binding to cytochrome P-450. The negative charge of the fatty acid was found to be involved in its association with the protein. Cytochrome P-450 was shown to interact only with a few spin-labeled phospholipid molecules in such a way that the motional restriction of the spin acyl chains can be detected by electron paramagnetic resonance (τR > 10?8s). The number of associated lipid molecules per protein probably is too small to form a complete shell around the protein. This lipid-protein interaction could be destroyed by the addition of sodium cholate, in contrast to the fatty acid-protein interaction.  相似文献   

6.
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 b5, NADH-cytochrome b5 reductase, and NADPH-cytochrome c reductase activities. Aniline hydroxylase and ethylmorphine N-demethylase activities could be reconstituted upon mixing the purified cytochrome with an NADPH-cytochrome c reductase preparation (purified by a detergent method) and phosphatidyl choline.  相似文献   

7.
Purified cytochrome P450SCC 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 P450SCC into liposomes. A maximum amount of heme was incorporated into liposomes at a molar ratio of phospholipid to the cytochrome of approx. 200. When P450SCC was incorporated into the dioleoylglycerophosphocholine liposomes by the cholate-filtration method, the P450SCC-containing liposomes 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, P450SCC was less stable than P450SCC in aqueous solution. Cholesterol or adrenodoxin markedly stabilized the liposomal P450SCC. Liposomal P450SCC 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 P450SCC was subjected to p-chloromercuriphenyl sulfonic acid treatment. This reagent destroyed the liposomal P450SCC. These results suggest that the heme is located in the proximity of the p-chloromercuriphenyl sulfonic acid reacting sites which are exposed to the surface, or located on the vincinity of polar heads of the membrane.  相似文献   

8.
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-450LMredO2Complex 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 b5 is added. The latter protein does not appear to function as an electron carrier in this process.  相似文献   

9.
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, t12 = 12 days, which represents mainly decay of tubular protein. The similarity between the major half-life of cytochrome P-450, t12 = 3.3 days, 17α-hydroxylase, t12 = 2.3 days and the C17–C20 lyase, t12 = 3.4 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, t12 = 4.5 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 0.1182 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.
Highly purified divalent and monovalent antibodies against cytochrome b5, anti-b5 immunoglobulin G (IG) and anti-b5 Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti-b5 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-b5 Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome b5 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   

11.
The oxidation of norbornane by a reconstituted liver cytochrome P-450 system affords exo- and endo-2-norborneol in a ratio of 3.4:1. The ratio of these products was found to be 0.76:1 when exo,exo,exo,exo-2,3,5,6-tetradueteronorbornane was oxidized. Analysis of the mass spectra of the products from the deuterated hydrocarbon showed that 25% of the exo-norborneol contained four deuterium atoms whereas 9% of the endo-norborneol contained three deuterium atoms. These results, which indicate a very large isotope effect (kHkD = 11.5±1) and a significant amount of epimerization for the hydroxylation of norbornane by cytochrome P-450, suggest an initial hydrogen abstraction to give a carbon radical intermediate.  相似文献   

12.
Cytochrome b5 was extracted and purified from beef liver by a detergent method (cytochrome d-b5). The hydrophilic moiety which carries the heme group (cytochrome t-b5) was prepared by trypsin action upon pure cytochrome d-b5.Single-shelled lecithin liposomes form complexes with cytochromes d-b5 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 t-b5 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 TM. i.e. when the aliphatic chains are in a crystalline state, and above TM, when the alipathic chain are in a fluid state.1H NMR spectra show that even at the maximum cytochrome d-b5 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   

13.
In flash-illuminated, oxygen-evolving spinach chloroplasts and green algae, a free radical transient has been observed with spectral parameters similar to those of Signal II (g ≈ 2.0045, ΔHpp ≈ 19 G). However, in contrast with ESR Signal II, the transient radical does not readily saturate even at microwave power levels of 200 mW. This species is formed most efficiently with “red” illumination (λ < 680 nm and occurs stoichiometrically in a 1 : 1 ratio with P-700+. The Photosystem II transient is formed in less than 100 μs and decays via first-order kinetics with a halftime of 400–900 μs. Additionally, the t12 for radical decay is temperature independent between 20 and 4 °C; however, below 4 °C the transient signal exhibits Arrhenius behavior with an activation energy of approx. 10 kcal · mol?1. Inhibition of electron transport through Photosystem II by o-phenanthroline, 3-(3,4-dichlorophenyl)-1,1-dimethylurea or reduced 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone suppresses the formation of the light-induced transient. At low concentrations (0.2 mM), 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone partially inhibits the free radical formation, however, the decay kinetics are unaltered. High concentrations of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (1–5 mM) restore both the transient signal and electron flow through Photosystem II. These findings suggest that this “quinoidal” type ESR transient functions as the physiological donor to the oxidized reaction center chlorophyll, P-680+.  相似文献   

14.
Peter Nicholls 《BBA》1976,430(1):13-29
1. Formate inhibits cytochrome c oxidase activity both in intact mitochondria and submitochondrial particles, and in isolated cytochrome aa3. 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 aa3 (a3+a33+) and in the half-reduced species (a2+a33+). Comparison with cyanide-induced spectral shifts, towards the red, indicates that formate and cyanide have opposite effects on the aa3 spectrum, both in the fully oxidized and the half-reduced states. The formate spectra provide a new method of obtaining the difference spectrum of a32+ minus a33+, free of the difficulties with cyanide (which induces marked high → low spin spectral shifts in cytochrome a33+) and azide (which induces peak shifts of cytochrome a2+ towards the blue in both α- and Soret regions).3. The rate of formate dissociation from cytochrome a2+a33+-HCOOH is faster than its rate of dissociation from a3+a33+-HCOOH, especially in the presence of cytochrome c. The Ki 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 c reductase activity is also inhibited by formate, in a reaction competitive with succinate and dependent on [formate]2.5. Formate inhibition of ascorbate plus N,N,N′,N′-tetramethyl-p-phenyl-enediamine 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 c 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 aa3 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   

15.
The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the β2-adrenergic mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, β12- or β2-, but not α1-, α2-, or β1-receptor-blocking this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine (α1, α2, β1, β2), isoproterenol (β1, β2) or terbutaline (β2) showed that also in normal rat kidney diamine oxidase activity is under the control of catecholamine2-receptors through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered β2-receptors coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney.  相似文献   

16.
125I-labelled α2-macroglobulin complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent Kd values calculated from displacement experiments were 7.9 · 10?8 M for α2-macroglobulin-thrombin and 8.5 · 10?8 M for α2-macroglobulin-plasmin. Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. α2-Macroglobulin itself bound also to hepatocytes, but the affinity of the α2-macroglobulin complexes was higher than that of the inhibitor alone, and α2-macroglobulin was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their α2-macroglobulin complexes. The α2-macroglobulin-thrombin complex competed with the α2-macroglobulin-plasmin complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for α2-macroglobulin-proteinase and antithrombin III-proteinase complexes are different.  相似文献   

17.
Mitochondrial ubiquinol-cytochrome c reductase complex contains small amounts of succinate dehydrogenase. Estimates from electrophoresis indicate there is one dehydrogenase per eight complexes. This dehydrogenase transfers electrons to the b-c1 complex poorly, as judged by low succinate-ubiquinone and succinate-cytochrome c reductase activities. Electron transfer to the b-c1 complex is restored by reconstitution of the complex with phospholipid. This phospholipid dependent restoration of electron transfer indicates that either reconstitutive activity of the dehydrogenase is preserved under conditions where electron transfer is absent, or that addition of phospholipid allows one dehydrogenase to transfer electrons to multiple b-c1 complexes.  相似文献   

18.
Changes in proton concentration during the binding of dioxygen, carbon monoxide, and for the exchange of dioxygen by carbon monoxide, at ferrous-cytochrome P-450cam were measured by direct titration. Insufficient proton release was observed to support protonation-deprotonation of an axial cysteinyl sulfur donor as a mechanism for generation of hyper spectra in only the carbonylated ferrous state. Measurement of the P12 value for CO binding as a function of pH (the carbon monoxide Bohr effect) confirms the direct titration data.  相似文献   

19.
The in vitro incorporation of cytochrome b5 into purified plasma membranes was investigated by biochemical and immunological methods. Plasma membrane preparations incorporated three times less cytochrome b5 than did microsomal preparations; 60% of this cytochrome b5 could not be reduced by the NADH-cytochrome b5 reductase and was considered as being bound to the plasma membrane. The morphological observations made after the immunochemical labeling of cytochrome b5 clearly showed a good but asymmetrical distribution of the ferritin labeling: only the inner face of the plasma membrane incorporated cytochrome b5. These results are discussed with respect to theories which concern the subcellular membrane relationships in the cell.  相似文献   

20.
Quercetin inhibited a dog kidney (Na+ + K+)-ATPase preparation without affecting Km for ATP or K0.5 for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the (Na+ + K+)-ATPase activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the Km for ATP and the K0.5 for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on Vmax, Km for substrate, and K0.5 for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations.  相似文献   

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