排序方式: 共有44条查询结果,搜索用时 0 毫秒
1.
2.
Purification and characterization of two isofunctional forms of NAD(P)H: quinone reductase from mouse liver 总被引:2,自引:0,他引:2
NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) is a widely distributed enzyme which promotes two-electron reductions of quinones and thereby protects cells against damage by reactive oxygen species generated during oxidative cycling of quinones and semiquinone radicals. Quinone reductase activity represents a minor component (about 0.006%) of mouse liver cytosolic proteins under basal (uninduced) conditions. Two isofunctional forms of this quinone reductase have been purified to homogeneity (1700-fold) in 30% yield from the liver cytosols of female CD-1 mice in which the enzymes were induced by administration of 2(3)-tert-butyl-4-hydroxyanisole. The purification involved ion exchange, hydrophobic, and affinity chromatographies. The two enzyme forms have been designated "hydrophilic" and "hydrophobic" based on the order of elution from phenyl-Sepharose. The more abundant hydrophilic form has been crystallized in the presence of FAD in the form of macroscopic tetragonal crystals. The two forms have similar isoelectric points (pI 9.2) and subunit molecular weights (Mr = 30,000) and probably exist as dimers in the native state. Purified preparations of the enzymes are equiactive with NADH and NADPH and show almost complete dependence on added FAD for catalytic activity. The Km values for FAD of the hydrophilic and hydrophobic forms are 2.72 and 1.72 nM, respectively. Their catalytic activities are the same and are remarkably high for nicotinamide nucleotide-linked dehydrogenases; maximum velocities (expressed per mg of pure enzyme) approach 4000 units/mg of protein under appropriate assay conditions. When menadione is the electron acceptor, the Km value for this quinone is very low (Km congruent to 2 microM). Both enzyme forms are potently inhibited by dicoumarol. Rabbit antisera against the hydrophilic quinone reductase precipitate quantitatively the entire quinone reductase activity of mouse liver cytosols obtained from animals maintained on a standard diet or those induced with 3-tert-butyl-4-hydroxyanisole. The quinone reductase activity of rat liver cytosols is also quantitatively precipitated by this antiserum. 相似文献
3.
Arrangement of subunit IV in beef heart cytochrome c oxidase probed by chemical labeling and protease digestion experiments 总被引:1,自引:0,他引:1
F Malatesta V Darley-Usmar C de Jong L J Prochaska R Bisson R A Capaldi G C Steffens G Buse 《Biochemistry》1983,22(19):4405-4411
The arrangement of subunit IV in beef heart cytochrome c oxidase has been explored by chemical labeling and protease digestion studies. This subunit has been purified from four samples of cytochrome c oxidase that had been reacted with N-(4-azido-2-nitrophenyl)-2-aminoethyl[35S]-sulfonate (NAP-taurine), diazobenzene[35S]sulfonate, 1-myristoyl-2-[12-[(4-azido-2-nitrophenyl)amino]lauroyl]-sn-glycero-3- [14C]phosphocholine (I), and 1-palmitoyl-2-(2-azido-4-nitrobenzoyl)-sn-glycero-3-[3H]phosphocholine (II), respectively. The labeled polypeptide was then fragmented by cyanogen bromide, at arginyl side chains with trypsin (after maleylation), and the distribution of the labeling within the sequence was analyzed. The N-terminal part of subunit IV (residues 1-71) was shown to be heavily labeled by water-soluble, lipid-insoluble reagents but not by the phospholipid derivatives. These latter reagents labeled only in the region of residues 62-122, containing the long hydrophobic and putative membrane-spanning stretch. Trypsin cleavage of native cytochrome c oxidase complex at pH 8.2 was shown to clip the first seven amino acids from subunit IV. This cleavage was found to occur in submitochondrial particles but not in mitochondria or mitoplasts. These results are interpreted to show that subunit IV is oriented with its N terminus on the matrix side of the mitochondrial inner membrane and spans the membrane with the extended sequence of hydrophobic lipid residues 79-98 buried in the bilayer. 相似文献
4.
Summary We have used a combination of chemical labeling and detergent fractionation techniques to locate the divalent cation binding sites on the chloroplast membrane. We determined the Ca2+-binding properties of Triton X-100 subchloroplast particles. Photosystem II (TSFII) particles showed one binding site withn=8.4 moles-mg chl–1 andk
d
=20 m. Photosystem I (TSFI) particles contained two binding sites. The first had ann=1.5 moles-mg chl–1 andk
d
=4 m. The second had ann=9.6 moles-mg chl–1 andk
d
=160 m. We have previously shown (Prochaska & Gross,Biochim. Biophys. Acta
376:126, 1975) that the divalent cation binding sites could be blocked using a water-soluble carbodiimide plus a nucleophile. Chlorophylla fluorescence and lightscattering changes were affected at the same carbodiimide concentrations emphasizing the relationship between these processes. The carbodiimide-sensitive sites were found to be located on the Photosystem II particles. A direct correlation between the inhibition of calcium binding and the carbodiimide-mediated incorporation of a (14C)-nucleophile was observed upon varying such parameters as carbodiimide concentration, nucleophile concentration, pH, and time of reaction. The presence of CaCl2 during the carbodiimide plus nucleophile modification procedure decreased the incorporation of (14C)-nucleophile, emphasizing the competition of the CaCl2 and the modification reagents for some of the same sites. Sodium dodecylsulfate gel electrophoresis of chlorophyll protein aggregates suggested that the site of competition of the calcium chloride and the modification reagents was the light-harvesting chlorophylla/b protein. 相似文献
5.
Beef heart cytochrome c oxidase has been reacted with [35S]diazobenzenesulfonate ([35S]DABS), [35S]-N-(4-azido-2-nitrophenyl)-2-aminoethylsulfonate ([35S]NAP-taurine), and two different radioactive arylazidophospholipids. The labeling of the seven different subunits of the enzyme with these protein modifying reagents has been examined. DABS, a water-soluble, lipid-insoluble reagent, reacted with subunits II, III, IV, V, and VII but labeled I or VI only poorly. The arylazidophospholipids, probes for the bilayer-intercalated portion of cytochrome c oxidase, labeled I, III, and VII heavily and II and IV lightly but did not react with V or VI. NAP-taurine labeled all of the subunits of cytochrome c oxidase. Evidence is presented that this latter reagent reacts with the enzyme from outside the bilayer, and the pattern of labeling with the different hydrophilic and hydrophobic labeling reagents is used to derive a model for the arrangement of subunits in cytochrome c oxidase. 相似文献
6.
Inhibition of cytochrome c oxidase function by dicyclohexylcarbodiimide 总被引:12,自引:0,他引:12
L J Prochaska R Bisson R A Capaldi G C Steffens G Buse 《Biochimica et biophysica acta》1981,637(2):360-373
Dicyclohexylcarbodiimide (DCCD) reacted with beef heart cytochrome c oxidase in inhibit the proton-pumping function of this enzyme and to a lesser extent to inhibit electron transfer. The modification of cytochrome c oxidase in detergent dispersion or in vesicular membranes was in subunits II-IV. Labelling followed by fragmentation studies showed that there is one major site of modification in subunit III. DCCD was also incorporated into several sites in subunit II and at least one site of subunit IV. The major site in subunit III has a specificity for DCCD at least one order of magnitude greater than that of other sites (in subunits II and IV). Its modification could account for all of the observed effects of the reagent, at least for low concentrations of DCCD. Labelling of subunit II by DCCD was blocked by prior covalent attachment of arylazidocytochrome c, a cytochrome c derivative which binds to the high-affinity binding site for the substrate. The major site of DCCD binding in subunit III was sequenced. The label was found in glutamic acid 90 which is in a sequence of eight amino acids remarkably similar to the DCCD-binding site within the proteolipid protein of the mitochondrial ATP synthetase. 相似文献
7.
The chemical modification reagents iodoacetic acid (primarily sulfhydryl group directed) and acetic anhydride (primarily amino group directed) were used to monitor chloroplast thylakoid membrane conformational changes. The incorporation of [3H]-iodoacetate and [3H]acetic anhydride showed the following pattern: (i) There was an increased level of binding of iodoacetate in the light compared to the dark or light plus 2,4-dichlorophenyl-1,1-dimethyl urea (DCMU) conditions. A 30 to 50% increase, from about 1.0 to 1.3–1.5 nmol/mg of Chl in iodoacetate incorporation, was found; 30–50% less acetic anhydride was bound in the light than in the dark or light plus DCMU state, typical values being near 15 nmol of acetic anhydride bound/mg of Chl in the dark and 10 nmol/mg of Chl in the light, (ii) The incorporation pattern for both reagents indicated that Photosystem II-dependent proton release is required to elicit the differential binding. Evidence for this is: (a) Cyclic electron flow and proton accumulation, mediated by phenazine methosulfate in the presence of a Photosystem II inhibitor (DCMU), did not induce either the extra binding of iodoacetate or the decrease in binding of acetic anhydride; (b) in chloroplasts made deficient in water oxidation by NH2OH treatment, electron flow from I?, an alternate Photosystem II electron donor, to methyl viologen did not induce the differential binding, whereas with the proton-donating donor, diphenyl carbazide, Photosystem II electron flow did elicit the differential binding, (iii) Uncouplers of phosphorylation (nigericin plus valinomycin) had no affect on the differential binding of either reagent, consistent with the hypothesis that it is not simply a transmembrane proton gradient that potentiates the conformational change, but rather an intramembrane reaction between protons released by Photosystem II and certain membrane components. The lack of uncoupler effect also suggests that the conformational change does not involve the coupling factor complex, at least not in the same sense as for the coupling factor conformational changes detected by tritium exchange (I. J. Ryrie and A. T. Jagendorf, 1971, J. Biol. Chem.246, 582–588) or N-ethyl maleimide binding (R. E. McCarty et al., 1972, J. Biol. Chem.247, 3048–3051). (iv) The decrease in acetic anhydride binding in the light was independent of the structural state of the chloroplast. Stacked and unstacked (by low salt) grana membranes showed similar light-dependent decreases in acetic anhydride binding. The results with these modification reagents support earlier conclusions about a Photosystem II-linked conformational change based on work with diazonium benzenesulfonic acid (R. Giaquinta et al., 1975, Biochemistry14, 4392–4396). 相似文献
8.
The low molecular weight (LMW) heat shock protein (HSP), HSP16.6, in the unicellular cyanobacterium, Synechocystis sp. PCC 6803, protects cells from elevated temperatures. A 95% reduction in the survival of mutant cells with an inactivated
hsp16.6 was observed after exposure for 1 h at 47°C. Wild-type cell survival was reduced to only 41%. HSP16.6 is also involved in
the development of thermotolerance. After a sublethal heat shock at 43°C for 1 h and subsequent challenge exposure at 49°C
for 40 min, mutant cells did not survive, while 64% of wild-type cells survived. Ultrastructural changes in the integrity
of thylakoid membranes of heat-shocked mutant cells also are discussed. These results demonstrate an important protective
role for HSP16.6 in the protection of cells and, in particular, thylakoid membrane against thermal stress.
Received: 14 October 1999 / Accepted: 16 November 1999 相似文献
9.
10.
Discontinuous sucrose gradient ultracentrifugation was used to separate liposomes containing Rhodobacter sphaeroides cytochrome c oxidase (pCOV) from liposomes devoid of the enzyme, and the biophysical and biochemical properties of pCOV were compared to unpurified liposomes containing cytochrome c oxidase (COV). Isolated and purified R. sphaeroides cytochrome c oxidase (COX) was reconstituted into asolectin phospholipid vesicles by cholate dialysis, and this preparation was purified further on a discontinuous sucrose gradient to isolate only those vesicles which contained the enzyme (pCOV). After centrifugation at 300,000g for 22h, 80% of the enzyme recovered was in a single band. The number of COX molecules per pCOV liposome was estimated by measuring the visible absorbance spectrum of cytochrome c oxidase (for heme aa(3)) and inorganic phosphate concentration (for phospholipid). The number of COX molecules incorporated per pCOV was estimated to be approximately one (0.72+/-0.19-1.09+/-0.28). The pCOV exhibited similar physical properties as COV; respiratory control ratios (indicators of endogenous proton permeability) and maximum enzymatic turnover number at pH 7.4 were comparable (6.0+/-1.3 and 535+/-130s(-1)). Furthermore, proton pumping activities of the pCOV were at least 70% of COV, indicating that discontinuous sucrose gradient centrifugation is a useful technique for functional experiments in R. sphaeroides cytochrome c oxidase. Our results suggest that the monomeric form of R. sphaeroides COX when reconstituted into a phospholipid bilayer is completely functionally active in its ability to perform electron transfer and proton pumping activities of the enzyme. 相似文献