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1.
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   

2.
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   

3.
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   

4.
According to previous authors, cytochrome b5, when extracted from bovine liver by a detergent method, is called cytochrome d-b5. On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome t-b5.Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, R, decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome d-b5, whereas R does not change for cytochrome c and cytochrome t-b5. This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome d-b5 occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome d-b5, because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature.  相似文献   

5.
The binding of cytochrome b5 to single-walled liposomes of egg phosphatidylcholine was inhibited by the presence of cholesterol in the lipid bilayer under conditions where a limited amount of liposomes was incubated with the cytochrome. Since similar conditions seem to apply for the binding of cytochrome b5 to erythrocyte ghosts, this observation supports the conclusion of Enomoto and Sato (Enomoto, K. and Sato, R. (1977) Biochim. Biophys. Acta 466, 136–147) that the localization of cholesterol on the outer surface of the ghost membrane prevents the binding of cytochrome b5 to this surface. The finding reported by Roseman et al. (Roseman, M.A., Holloway, P.W. and Calabro, M.A. (1978) Biochim. Biophys. Acta 507, 552–556) that cholesterol did not prevent the cytochrome binding to phosphatidylcholine liposomes in the presence of a large excess of liposomes could be confirmed in the present study, but this does not contradict the abovementioned conclusion.  相似文献   

6.
The kinetics of pyruvate transport across the isolated red blood cell membrane were studied by a simple and precise spectrophotometric method: following the oxidation of NADH via lactate dehydrogenase trapped within resealed ghosts. The initial rate of pyruvate entry was linear. Influx was limited by saturation at high pyruvate concentration. Pyruvate influx was greatly stimulated by increasing ionic strength in the outer but not the inner aqueous compartment. The Km ranged from 15.0 mM at μ = 0.05 to 3.7 mM at μ = 0.01, while the V went from 0.611 · 10-15 to 0.137 · 10-15mol · min-1 · ghost-1. Ionic strength was shown to affect the translocation step and not pyruvate binding. The energy of activation of pyruvate flux into resealed ghosts was 25 kcal/mol, similar to that found in intact red blood cells. Inhibitors of pyruvate influx included such anions as thiocyanate, chloride, bicarbonate, α-cyanocinnamate, salicylate and ketomalonate (but not acetate); noncompetitive inhibitors were phloretin, 1-fluoro-2,4-dinitrobenzene, 4-acetamido-4′-isothiocyanate-stilbene-2,2′-disulfonic acid and o-phenanthroline/CuSO4 mixtures. The last reagent, known to induce disulfide links in certain membrane proteins, blocked the ionic strength stimulation of pyruvate influx in this study.  相似文献   

7.
DNA complementary to rabbit globin mRNA made by E. coli polymerase I   总被引:2,自引:0,他引:2  
Incubation of liver microsomes with cytochrome b5, purified after solubilization with detergents, caused an effective incorporation of the cytochrome into the microsomal membranes. The incorporated cytochrome was reducible by NADH and could not be removed by repeated washing with 0.3 M KCl or 10 mM EDTA. The incorporation was much more efficient at 37°C than at 0°C. Trypsin-solubilized cytochrome b5, which lacks the hydrophobic tail of the native protein, could not be inserted into the membranes. These findings confirm the view that the hydrophobic tail of the cytochrome molecule is responsible for its tight binding to the microsomal membranes.  相似文献   

8.
The distribution and site of synthesis of cytochrome b5 was studied by antibody precipitation of the enzyme labeled invivo. The enzyme is present in rough and smooth microsomes, Golgi and outer mitochondrial membranes. The cytochrome is synthesized only on bound ribosomes, where glucosamine and galactose moieties are also added. The enzyme seems to be devoid of mannose and sialic acid residues.  相似文献   

9.
The lateral diffusion of the excimer-forming probe pyrene decanoic acid has been determined in erythrocyte membranes and in vesicles of the lipid extracts. The random walk of the probe molecules is characterized by their jump frequency, vj, within the lipid matrix. At T = 35°C a value of vj = 1.6 · 103 s?1 is found in erythrocyte membranes. A somewhat slower mobility is determined in vesicles prepared from lipid extracts of the erythrocyte membrane. Depending on structure and charge of the lipids we obtain jump frequencies between 0.8 · 108 s?1 and 1.5 · 108 s?1 at T = 35°C. The results are compared with jump frequencies yielded in model membranes.The mobility of molecules perpendicular to the membrane surface (transversal diffusion) is investigated. Erythrocyte ghosts doped with pyrene phosphatidylcholine were mixed with undoped ghosts in order to study the exchange kinetics of the probe molecule. A fast transfer between the outer layers of the ghost cells (τ12 = 1.6 min at T = 37°C) is found. The exchange process between the inner and the outer layer of one erythrocyte ghost (flip-flop process) following this fast transfer occurs with a half-life time value of t12 = 100 min at T = 37°C.The application of excimer-forming probes presumes a fluid state of the membrane. Therefore we investigated the phase transition behaviour using the excimer technique. Beside a thermotropic phase transition at T = 23°C and T = 33°C we observed an additional fluidity change at T = 38°C in erythrocyte ghosts. This transition is attached to a separation of the boundary lipid layer from the intrinsic proteins. No lipid phase transition is observed in liposomes from isolated extracts of the erythrocyte membrane with our methods.  相似文献   

10.
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   

11.
[N-13CH3] Phosphatidylcholines are introduced into the outer monolayer of phosphatidylcholine vesicles with the phosphatidylcholine exchange protein from bovine liver. The transbilayer distribution of the [N-13CH3] phosphatidylcholine is measured with 13C NMR. The transbilayer movements of [N-13CH3]-dioleoyl phosphatidylcholine and [N-13CH3] dimyristoyl phosphatidylcholine at 30°C in vesicles composed of these phosphatidylcholines are extremely slow processes with estimated half-times of days. [N-13CH3] Dioleoyl phosphatidylcholine introduced into dimyristoyl phosphatidylcholine vesicles migrates from the outer to the inner monolayer with a half-time of less than 12 h. The data suggest that differential changes in the lateral packing of the two monolayers might be a driving force for transbilayer transport of phospholipids.  相似文献   

12.
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   

13.
14.
The cytochrome b5b5 reductase system solubilized from microsomes exhibits monophasic reduction kinetics over the temperature range 15 ° to ?25 °C in aqueous/ethylene glycol co-solvent, whereas in intact microsomes, the process becomes increasingly heterogeneous below 0 °C, reflecting heterogeneities in membrane structure observable as distributions in reaction rates and activation energies.  相似文献   

15.
A protease which generates a soluble hemepeptide from bovine liver microsomal cytochrome b5 has been isolated from the membrane fraction of rabbit reticulocytes. Inhibition by pepstatin and an acidic pH optimum indicate that the protease belongs to the acid protease class. Little cytochrome b5-processing activity is observed in rabbit erythrocytes. We suggest that the protease may be involved in the processing which generates the proteins of the methemoglobin reduction system from their membrane-bound precursors during the maturation of the erythroid cell.  相似文献   

16.
Quenching of the fluorescence of the (Ca2+ + Mg2+)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.  相似文献   

17.
A protein named oxidation factor can be reversibly removed from succinate-cytochrome c reductase complex and shown to be required for electron transfer between succinate and cytochrome c. This protein is required for reduction of cytochrome c1 and, in the presence of antimycin, for reduction of both cytochromes b and c1. These results are consistent with a protonmotive Q cycle mechanism in which the oxidation factor catalyzes electron transfer from reduced quinone to cytochrome c1 and thus liberates from reduced quinone one of two protons required for energy conservation during electron transfer through the cytochrome b-c1 complex.  相似文献   

18.
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   

19.
The phase transition temperature (Tt) of dipalmitoyl phosphatidic acid multilamellar liposomes is depressed 10°C by the inhalation anesthetic methoxyflurane at a concentration of 100 mmol/mol lipid. Application of 100 atm of helium pressure to pure phosphatidic acid liposomes increased Tt only 1.5°C. However, application of 100 atm helium pressure to dipalmitoyl phosphatidic acid lipsomes containing 100 mmol methoxyflurane/mol lipid almost completely antagonized the effect of the anesthetic. A nonlinear pressure effect is observed. In a previous study, a concentration of 60 mmol methoxyflurane/mol dipalmitoyl phosphatidylcholine depressed Tt only 1.5°C, exhibiting a linear pressure effect. The completely different behavior in the charged membrane is best explained by extrusion of the anesthetic from the lipid phase.  相似文献   

20.
A green mutant of Rhodopseudomonas spheroides was isolated in which spectroscopic measurements of the α-band region of cytochromes could be made. It was grown either aerobically or photosynthetically, and the membrane fractions prepared from cells of each type. Anaerobic potentiometric titration at 560 nm minus 542 nm showed the same three redox components, tentatively identified as b-type cytochromes, in membrane fractions from either type of cell. The mid-point potentials were approximately +185, +41 and ?104 mV. In membranes from photosynthetically grown cells the major cytochrome form absorbing at 560 nm had a mid-point potential of +42 mV; in aerobically grown cells the major form had a potential of +185 mV. In both types of cell only one c-type cytochrome was found, with a mid-point potential of +295 mV. An a-type cytochrome was present only in aerobically-grown cells.Substrate-reduced particles from these cells were mixed with air-saturated buffer in a stopped-flow spectrophotometer and the kinetics of oxidation of b- and c-type cytochromes were measured. The same two b-type components, reacting with pseudo first order kinetics, were detected in particles from both aerobically and photosynthetically grown cells (t12 for oxidation 1.3 s and 0.13 s). The c-type cytochrome of particles from aerobically grown cells was oxidised with t12 of 0.97 s; the c-type cytochrome of photosynthetic cells was oxidised faster, with t12 of 0.27 s.These observations have implications on the adaptive formation of electron transport systems that are discussed.  相似文献   

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