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1.
Hen liver microsomes contained 0.20 nmol of cytochromeb5 per mg of protein. Upon addition of NADH about 95% cytochrome b5 was reduced very fast with a rate constant of 206 s?1When ferricyanide was added to the reaction system the cytochrome stayed in the oxidized form until the ferricyanide reduction was almost completed. The reduced cytochrome b5 in microsomes was oxidized very rapidly by ferricyanide. The rate constant of 4.5 × 108m?1 s?1, calculated on the basis of assumption that ferricyanide reacts directly with the cytochrome, was found to be more than 100 times higher than that of the reaction between ferricyanide and soluble cytochrome b5. To explain the results, therefore, the reverse electron flow from cytochrome b5 to the flavin coenzyme in microsomes was assumed.By three independent methods the specific activity of the microsomes was measured at about 20 nmol of NADH oxidized per s per mg of protein and it was concluded that the reduction of the flavin coenzyme of cytochrome b5 reductase by NADH is rate-limiting in the NADH-cytochrome b5 and NADH-ferricyanide reductase reactions of hen liver microsomes. In the NADH-ferricyanide reductase reaction the apparent Michaelis constant for NADH was 2.8 μm and that for ferricyanide was too low to be measured. In the NADH-cytochrome c reductase reaction the maximum velocity was 2.86 nmol of cytochrome c reduced per s per mg of protein and the apparent Michaelis constant for cytochrome c was 3.8 μm. 相似文献
2.
《Insect Biochemistry》1979,9(3):301-308
The cytochrome b5-like protein in the microsomal fraction of southern armyworm (Spodoptera eridania) larval midguts was solubilized with bromelain and purified about 130-fold to a specific content of 55.4 nmoles per mg protein. The protein had protoheme as its prosthetic group, a molecular weight of 11,400 ± 600 and its amino acid content and spectral properties were similar to those of cytochrome b5 purified from rat liver microsomes by a similar procedure. The midpoint oxidation-reduction potential (E0′) was −57 mV. The results strongly suggest that the armyworm midgut cytochrome is a structurally similar protein to mammalian cytochrome b5. 相似文献
3.
The incorporation of radioactive amino acids and of δ-amino[2,3-3H2]laevulinate into rat liver cytochromes b5 and c and cytochrome oxidase has been examined with and without protein-synthesis inhibitors. Cycloheximide promptly inhibits labelling of both haem and protein for cytochrome c in parallel fashion. Although incorporation of 14C-labelled amino acid into microsomal cytochrome b5 is also rapidly inhibited, cycloheximide incompletely inhibits haem labelling of cytochrome b5 and cytochrome a+a3, and inhibition occurs only after repeated antibiotic injections. The possibility of apo-protein pools, or of haem exchange, with a rapidly renewed `free' haem pool, is considered. Consistent with this model is the observation of non-enzymic haem exchange in vitro between cytochrome b5 and methaemoglobin. Chloramphenicol, injected intravenously over 5h, results in a 20–40% decrease in incorporation of δ-amino[2,3-3H2]laevulinate into haem a+a3 and haem of cytochromes b5 and c. With the dosage schedule of chloramphenicol studied, amino acid labelling of total liver protein and of cytochrome c was not inhibited. Similarly, ferrochelatase activity was not decreased. 相似文献
4.
Cytochrome b5 purified from neonatal pig testis and that from pig liver stimulated C21 steroid side-chain cleavage (progesterone → androstenedione) catalyzed in vitro by purified cytochrome P-450 from neonatal pig testicular microsomes. Km of testicular cytochrome b5 for the P-450 is 6.3–9.1 × 10?8M and the ability of b5 to stimulate C21 side-chain cleavage is different for cytochromes b5 prepared from different sources. 相似文献
5.
(i) Compounds activating the microsomal electron transfer oxidative reactions, e.g., the mixed function oxidase (aminopyrine, aniline), the Δ9-desaturase (stearyl-CoA), and lipid peroxidation reaction (iron pyrophosphate), cause a decrease in the steady-state reduced level of cytochrome b5. (ii) In the absence of substrates, the kox for cytochrome b5 was the same whether reduced by NADH or NADPH (about 0.045 S?1, indicating that no distinction exists between the cytochrome b5 involved in NADH-driven and NADPH-driven microsomal reactions which utilize this hemoprotein. (iii) The agents activating the oxidative pathways affect the first-order rate constant for cytochrome b5 oxidation (kox), but the apparent first-order rate constant obtained for reduction (kred) of cytochrome b5 by NADPH is still more than 10 times the kox, and the kred obtained with NADH is still more than 100 times the kox. (iv) Of the compounds used, only stearyl-CoA caused a decrease in the NADH-supported steady-state reduced level of cytochrome b5. This effect is probably due to a detergent-like action of stearyl-CoA on the membrane proteins, interfering with some interactions (e.g., NADPH-cytochrome c reductase with cytochrome P-450; NADH-cytochrome b5 reductase with cytochrome b5). (v) Based upon the kinetic and steady-state measurements it is concluded that substrate-induced changes in the steady-state reduced level of cytochrome b5 are evidence for a decrease in the population of this hemoprotein available to the reductase due to competition with other more favored acceptors, (vi) Measurements using the duration of the reduced state and rates of electron flow through cytochrome b5 reveal that normally about 60% of the NADH-derived reducing equivalents go through cytochrome b5 while only about one electron in nine passes through this cytochrome when NADPH is the source of reducing equivalents. Substrates of the various pathways alter the proportion of electrons passing through cytochrome b5 depending upon their activating or inhibiting action on cytochrome b5-dependent or -independent reactions. 相似文献
6.
Alejandro K. Samhan-Arias Rui M. Almeida Susana Ramos Cristina M. Cordas Isabel Moura Carlos Gutierrez-Merino José J.G. Moura 《BBA》2018,1859(2):78-87
Cytochrome b5 is the main electron acceptor of cytochrome b5 reductase. The interacting domain between both human proteins has been unidentified up to date and very little is known about its redox properties modulation upon complex formation. In this article, we characterized the protein/protein interacting interface by solution NMR and molecular docking. In addition, upon complex formation, we measured an increase of cytochrome b5 reductase flavin autofluorescence that was dependent upon the presence of cytochrome b5. Data analysis of these results allowed us to calculate a dissociation constant value between proteins of 0.5 ± 0.1 μM and a 1:1 stoichiometry for the complex formation. In addition, a 30 mV negative shift of cytochrome b5 reductase redox potential in presence of cytochrome b5 was also measured. These experiments suggest that the FAD group of cytochrome b5 reductase increase its solvent exposition upon complex formation promoting an efficient electron transfer between the proteins. 相似文献
7.
The influence of charged phospholipid membranes on the conformational state of the water-soluble fragment of cytochrome b5 has been investigated by a variety of techniques at neutral pH. The results of this work provide the first evidence that aqueous solutions with high phospholipid/protein molar ratios (pH 7.2) induce the cytochrome to undergo a structural transition from the native conformation to an intermediate state with molten-globule like properties that occur in the presence of an artificial membrane surface and that leads to binding of the protein to the membrane. At other phospholipid/protein ratios, equilibrium was observed between cytochrome free in solution and cytochrome bound to the surface of vesicles. Inhibition of protein binding to the vesicles with increasing ionic strength indicated for the most part an electrostatic contribution to the stability of cytochrome b5vesicle interactions at pH 7.2. The possible physiological role of membrane-induced conformational change in the structure of cytochrome b5 upon the interaction with its redox partners is discussed. 相似文献
8.
Lipid-protein films at the air-water interface were generated from a variety of native vesicles and from vesicles derived from lipid extracts. A technique is described which is particularly suitable for the generation of films from small amounts of material at high yield and velocity. In all instances, 10 l vesicle suspensions containing 25 g protein yield at least 50 cm2 film area at a constant surface pressure of 12 mN/m within minutes. Upon formation, surface films are separated from vesicles by use of shear forces. Complete separation is demonstrated by electron microscopy and surface pressure-area diagrams. The latter confirms previous conclusions that surface films generated from lipid vesicles are organized as a monolayer. Analysis of lipid-protein surface layers reveals that their lipid to protein ratios match those of the vesicles used, within a factor of two, irrespective of whether films are generated at high or low surface pressure. Surface denaturation of membrane proteins is shown to be effectively prevented when the film is generated and held at high surface pressure ( 15 mN/m). Upon surface pressure jumps from high to low values, denaturation kinetics revealed activation areas of 1.5 (±0.2) nm2.
Offprint requests to: H. Schindler 相似文献
9.
《Journal of steroid biochemistry》1980,12(8):861-867
Addition of pregnenolone to guinea-pig adrenal microsomes produces a slowly developing difference spectrum with peaks at about 425 and 557 nm and a trough at about 410 nm. The spectral change is similar to that resulting from the reduction of cytochrome b5 by NADH or NADPH. In the presence of sufficient quantities of NADH to fully reduce cytochrome b5, pregnenolone produces a typical type I difference spectrum (ΔA385–420 nm). Pregnenolone is converted to progesterone by adrenal microsomes without addition of cofactor (NAD+) for the 3β-hydroxysteroid dehydrogenase (HSD) reaction. The rate of conversion is increased 2–3 fold by NAD+ and inhibited by NADH. Accompanying the metabolism of pregnenolone (with or without added NAD+) is the production of NADH and reduction of cytochrome b5. Addition of pregnenolone alone to adrenal microsomes results in 60–80% reduction of cytochrome b5. The reduction of cytochrome b5 is maintained for at least as long as pregnenolone is being metabolized. Inhibition of pregnenolone metabolism changes the pregnenolone-induced spectral change to a type I and prevents the reduction of cytochrome b5. The results suggest that the oxidation-reduction state of cytochrome b5 in adrenal microsomes is controlled in part by pregnenolone metabolism which in turn influences the pregnenolone-induced difference spectrum. Oxidation of NADH by cytochrome b5 may serve to prevent NADH inhibition of HSD activity and to generate additional NAD+ as cofactor for the reaction. 相似文献
10.
《Biophysical journal》2022,121(18):3533-3541
Cholesterol induces faster collapse by compressed films of pulmonary surfactant. Because collapse prevents films from reaching the high surface pressures achieved in the alveolus, most therapeutic surfactants remove or omit cholesterol. The studies here determined the structural changes by which cholesterol causes faster collapse by films of dipalmitoyl phosphatidylcholine, used as a simple model for the functional alveolar film. Measurements of isobaric collapse, with surface pressure held constant at 52 mN/m, showed that cholesterol had little effect until the mol fraction of cholesterol, Xchol, exceeded 0.20. Structural measurements of grazing incidence X-ray diffraction at ambient laboratory temperatures and a surface pressure of 44 mN/m, just below the onset of collapse, showed that the major structural change in an ordered phase occurred at lower Xchol. A centered rectangular unit cell with tilted chains converted to an untilted hexagonal structure over the range of Xchol = 0.0–0.1. For Xchol = 0.1–0.4, the ordered structure was nearly invariant; the hexagonal unit cell persisted, and the spacing of the chains was essentially unchanged. That invariance strongly suggests that above Xchol = 0.1, cholesterol partitions into a disordered phase, which coexists with the ordered domains. The phase rule requires that for a binary film with coexisting phases, the stoichiometries of the ordered and disordered regions must remain constant. Added cholesterol must increase the area of the disordered phase at the expense of the ordered regions. X-ray scattering from dipalmitoyl phosphatidylcholine/cholesterol fit with that prediction. The data also show a progressive decrease in the size of crystalline domains. Our results suggest that cholesterol promotes adsorption not by altering the unit cell of the ordered phase but by decreasing both its total area and the size of individual crystallites. 相似文献
11.
The physiological transient complex between cytochrome f (Cf) and cytochrome c6 (Cc6) from the cyanobacterium Nostoc sp. PCC 7119 has been analysed by NMR spectroscopy. The binding constant at low ionic strength is 8 ± 2 mM−1, and the binding site of Cc6 for Cf is localized around its exposed haem edge. On the basis of the experimental data, the resulting docking simulations suggest that Cc6 binds to Cf in a fashion that is analogous to that of plastocyanin but differs between prokaryotes and eukaryotes. 相似文献
12.
The Structure of Calf Liver Cytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>5</Subscript> at 2.8 Å Resolution 总被引:5,自引:0,他引:5
CYTOCHROME b5 is a haem-containing protein in the microsomes of liver tissue. It interacts specifically with a flavo-protein, cytochrome b5 reductase, which catalyses the transfer of electrons from NADH to the haem iron of the cytochrome1. The microsomal cytochrome b5 system has been implicated in fatty acid desaturation reactions2 and a similar system in erythrocytes may catalyse the reduction of methaemoglobin3. Calf liver cytochrome b5, solubilized by pancreatic lipase, has a molecular weight of 11,000 and consists of ninety-three amino-acids in the sequence shown in Fig. 1 (refs. 4 and 5). The haem group is non-covalently bound to the protein and can be removed reversibly by acid acetone treatment6. 相似文献
13.
Electrostatic properties on the protein surface were examined on the basis of the crystal structure of NADH-cytochrome b5 reductase refined to a crystallographic R factor of 0.223 at 2.1 Å resolution and of the other three flavin-dependent reductases. A structural comparison of NADH-cytochrome b5 reductase with the other flavin-dependent reductases, ferredoxin-NADP+ reductase, phthalate dioxygenase reductase, and nitrate reductase, showed that the α/β structure is the common motif for binding pyridine nucleotide. Although the amino acid residues associated with pyridine nucleotide-binding are not conserved, the electrostatic properties and the location of the pyridine nucleotide-binding pockets of NADH-requiring reductases were similar to each other. The electrostatic potential of the surface near the flavin-protruding side (dimethylbenzene end of the flavin ring) of NADH-cytochrome b5 reductase was positive over a wide area while that of the surface near the heme-binding site of cytochrome b5 was negative. This implied that the flavin-protruding side of NADH-cytochrome b5 reductase is suitable for interacting with its electron-transfer partner, cytochrome b5. This positive potential area is conserved among four flavin-dependent reductases. A comparison of the electron-transfer partners of four flavin-dependent reductases showed that there are significant differences in the distribution of electrostatic potential between inter-molecular and inter-domain electron-transfer reactions. © 1996 Wiley-Liss, Inc. 相似文献
14.
《Biochimica et Biophysica Acta - Proteins and Proteomics》2018,1866(2):373-378
In alkaline media (pH 12) a catalytic peroxidase activity of cytochrome b5 was found associated to a different conformational state. Upon incubation at this pH, cytochrome b5 electronic absorption spectrum was altered, with disappearance of characteristic bands of cytochrome b5 at pH 7.0. The appearance of new electronic absorption bands and EPR measurements support the formation of a cytochrome b5 class B hemichrome with an acquired ability to bind polar ligands. This hemichrome is characterized by a negative formal redox potential and the same folding properties than cytochrome b5 at pH 7. The acquired peroxidase-like activity of cytochrome b5 found at pH 12, driven by a hemichrome formation, suggests a role of this protein in peroxidation products propagation. 相似文献
15.
An antibody preparation elicited against purified, lysosomal-solubilized NADH-cytochrome b5 reductase from rat liver microsomes was shown to interact with methemoglobin reductase of human erythrocytes by inhibiting the rate of erythrocyte cytochrome b5 reduction by NADH. The ferricyanide reductase activity of the enzyme was not inhibited by the antibody, suggesting that the inhibition of methemoglobin reductase activity may be due to interference with the binding of cytochrorme b5 to the flavoprotein. Under conditions of limiting concentrations of flavoprotein, the antibody inhibited the rate of methemoglobin reduction in a reconstituted system consisting of homogeneous methemoglobin reductase and cytochrome b5 from human erythrocytes. This inhibition was due to the decreased level of reduced cytochrome b5 during the steady state of methemoglobin reduction while the rate of methemoglobin reduction per reduced cytochrome b5 stayed constant, suggesting that the enzyme was not concerned with an electron transport between the reduced cytochrome b5 and methemoglobin.An antibody to purified, trypsin-solubilized cytochrome b5 from rat liver microsomes was shown to inhibit erythrocyte cytochrome b5 reduction by methemoglobin reductase and NADH to a lesser extent than microsomal cytochrome b5 preparations from rat liver (trypsin solubilized or detergent solubilized) and pig liver (trypsin solubilized). The results presented establish that soluble methemoglobin reductase and cytochrome b5 of human erythrocytes are immunochemically similar to NADH-cytochrome b5 reductase and cytochrome b5 of liver microsomes, respectively. 相似文献
16.
《Insect Biochemistry》1989,19(5):481-488
Cytochrome P-450, cytochrome b5 and cytochrome P-450 reductase were purified from house fly abdomens using high performance liquid chromatography (HPLC). Using a new technique, cytochrome P-450 was separated from the bulk of other proteins after polyethylene glycol fractionation and hydrophobic interaction chromatography (HIC) using a phenyl-5PW column. This technique resulted in 91% recovery of the cytochrome P-450s in a single concentrated fraction that also contained the remaining cytochrome b5 and cytochrome P-450 reductase activity. Further purification by anion exchange on a DEAE-5SW column resolved the cytochrome P-450s, cytochrome b5 and cytochrome P-450 reductase into individual fractions. The ion exchange step yielded one fraction that contained a high specific content of P-450 (14.4 nmol/mg protein). This cytochrome P-450 fraction ran as a single band at 54.3 kDa in sodium dodecyl sulfate polyacrylamide (SDS-PAGE) gel electrophoresis and had a carboxy ferrocytochrome absorbance maximum at 447 nm.Further purification of the anion exchange cytochrome b5 fraction, by C8 reverse phase HPLC, resulted in a cytochrome b5 fraction with a specific content of 51.8 nmol/mg protein and an apparent molecular mass of 19.7 kDa by SDS-PAGE. The anion exchange HPLC fraction containing the cytochrome P-450 reductase activity was further purified by NADP-agarose affinity chromatography. This step yielded cytochrome P-450 reductase with an apparent molecular mass of 72 kDa. 相似文献
17.
Shigetaka Kitajima Yukio Yasukochi Shigeki Minakami 《Archives of biochemistry and biophysics》1981,210(1):330-339
An NADH:(acceptor) oxidoreductase (EC 1.6.99.3) of human erythrocyte membrane was purified by DEAE-cellulose anion exchange, hydroxyapatite adsorption, and 5′-ADP-hexane-agarose affinity chromatographies after solubilization with Triton X-100. The purified reductase preparation was homogeneous and estimated to have an apparent molecular weight of 36,000 on SDS-polyacrylamide slab gel electrophoresis and of 144,000 on Sephadex G-200 gel filtration in the presence of 0.2% Triton X-100, whereas a soluble NADH-cytochrome b5 reductase of human erythrocyte had a molecular weight of 32,000 by both methods, indicating the existence of a distinct membrane reductase. Digestion of the membrane reductase with cathepsin D yielded a new polypeptide chain which gave the same relative mobility as the soluble reductase on SDS-polyacrylamide slab gel electrophoresis. The membrane enzyme, the cathepsin-digested enzyme, and the soluble enzyme all cross-reacted with the antibody to rat liver microsomal NADH-cytochrome b5 reductase. The enzyme had one mole FAD per 36,000 as a prosthetic group and could reduce K3Fe(CN)6, 2,6-dichlorophenolindophenol, cytochrome c, methemoglobin-ferrocyanide complex, cytochrome b5 and methemoglobin via cytochrome b5 when NADH was used as an electron donor. NADPH was less effective as an electron donor than NADH. The specific activity of the purified enzyme was 790 μmol ferricyanide reduced min?1 mg?1 and the turnover number was 40,600 mol ferricyanide reduced min?1 mol?1 FAD at 25 °C. The apparent Km values for NADH and cytochrome b5 were 0.6 and 20 μm, respectively, and the apparent V value was 270 μmol cytochrome b5 reduced min?1 mg?1. These kinetic properties were similar to those of the soluble NADH-cytochrome b5 reductase. The results indicate that the NADH:(acceptor) oxidoreductase of human erythrocyte membrane could be characterized as a membrane NADH-cytochrome b5 reductase. 相似文献
18.
Serratos IN Pérez-Hernández G Garza-Ramos G Hernández-Arana A González-Mondragón E Zubillaga RA 《Journal of molecular biology》2011,405(1):158-172
Electrostatic interactions have a central role in some biological processes, such as recognition of charged ligands by proteins. We characterized the binding energetics of yeast triosephosphate isomerase (TIM) with phosphorylated inhibitors 2-phosphoglycollate (2PG) and phosphoglycolohydroxamate (PGH). We determined the thermodynamic parameters of the binding process (Kb, ΔGb, ΔHb, ΔSb and ΔCp) with different concentrations of NaCl, using fluorimetric and calorimetric titrations in the conventional mode of ITC and a novel method, multithermal titration calorimetry (MTC), which enabled us to measure ΔCp in a single experiment. We ruled out specific interactions of Na+ and Cl- with the native enzyme and did not detect significant linked protonation effects upon the binding of inhibitors. Increasing ionic strength (I) caused Kb, ΔGb and ΔHb to become less favorable, while ΔSb became less unfavorable. From the variation of Kb with I, we determined the electrostatic contribution of TIM−2PG and TIM−PGH to ΔGb at I = 0.06 M and 25 °C to be 36% and 26%, respectively. The greater affinity of PGH for TIM is due to a more favorable ΔHb compared to 2PG (by 19-24 kJ mol-1 at 25 °C). This difference is compatible with PGH establishing up to five more hydrogen bonds with TIM. Both binding ΔCps were negative, and less negative with increasing ionic strength. ΔCps at I = 0.06 M were much more negative than predicted by surface area models. Water molecules trapped in the interface when ligands bind to protein could explain the highly negative ΔCps. Thermodynamic binding functions for TIM−2PG changed more with ionic strength than those for TIM−PGH. This greater dependence is consistent with linked, but compensated, protonation equilibriums yielding the dianionic species of 2PG that binds to TIM, process that is not required for PGH. 相似文献
19.
The routes of microsomal electron flow to the three terminal oxidative enzymes, the mixed function oxidase, the fatty acyl CoA desaturase, and the lipid peroxidase have been examined by the use of specific antibodies, by alteration of electron transfer enzyme levels, and with the inhibitor NADP+. From these studies a number of conclusions are drawn: (1) NADH-supported lipid peroxidation utilizes NADH-cytochrome b5 reductase, but electron flow does not go via cytochrome b5. (2) The positive modifier effect of type I substrates on NADPH-driven cytochrome P-450 reduction is seen also with NADH-supported cytochrome P-450 reductase activity. The latter reaction proceeds via cytochrome b5 while the former does not. (3) Cross-reactivity can occur between NADH-cytochrome b5 reductase and NADPH-cytochrome c reductase, but at a rate too slow to support most reactions. (4) Cytochrome b5 appears to exist in two pools; one pool is readily inhibited by antibody and the other pool is either inaccessible to or incompletely inhibited by antibody. The various cytochrome b5-dependent reactions show different abilities to use the noninhibited hemoprotein. NADH-cytochrome c reductase activity and NADH-synergism appear to utilize only the former pool and are completely inhibitable by antibody. Other NADH-supported reactions (Δ9-desaturation and mixedfunction oxidation) utilize the total cytochrome b5 population. Fortification studies show that the extra bound cytochrome b5 is distributed in the same manner as the endogenous cytochrome b5. 相似文献
20.
Jeremy L. Dahmen Rebecca Olsen Deirdre Fahy James G. Wallis John Browse 《Eukaryotic cell》2013,12(6):923-931
Very-long-chain polyunsaturated fatty acids such as arachidonic, eicosapentaenoic, and docosahexaenoic acids, are important to the physiology of many microorganisms and metazoans and are vital to human development and health. The production of these and related fatty acids depends on Δ6 desaturases, the final components of an electron transfer chain that introduces double bonds into 18-carbon fatty acid chains. When a Δ6 desaturase identified from the ciliated protist Tetrahymena thermophila was expressed in Saccharomyces cerevisiae cultures supplemented with the 18:2Δ9,12 substrate, only 4% of the incorporated substrate was desaturated. Cytochrome b5 protein sequences identified from the genome of T. thermophila included one sequence with two conserved cytochrome b5 domains. Desaturation by the Δ6 enzyme increased as much as 10-fold when T. thermophila cytochrome b5s were coexpressed with the desaturase. Coexpression of a cytochrome b5 from Arabidopsis thaliana with the Δ6 enzyme also increased desaturation. A split ubiquitin growth assay indicated that the strength of interaction between cytochrome b5 proteins and the desaturase plays a vital role in fatty acid desaturase activity, illustrating the importance of protein-protein interactions in this enzyme activity. 相似文献