Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.9.3.1 (cytochrome oxidase)
8,822 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Evidence for the presence of a quinol oxidase super-complex composed of a cytochrome bc1 complex and cytochrome oxidase in the respiratory chain of a Gram-positive thermophilic bacterium PS3 is reported. On incubation with an octyl glucoside-solubilized fraction of the total membranes of PS3 anti-serum against PS3 cytochrome oxidase gave an immunoprecipitate that showed both quinol-cytochrome c reductase and cytochrome c oxidase activities. When the cholate-deoxycholate and LiCl-treated membranes of PS3 were solubilized and subjected to ion-exchange chromatography in the presence of octaethyleneglycol dodecyl ether, most of the A-, B-, and C-type cytochromes were copurified as a peak having both quinol-cytochrome c reductase and cytochrome oxidase activities. The immunoprecipitate and quinol oxidase preparation contained hemes a, b, and c in a ratio of about 2:2:3, indicating the presence of one-to-one complex of cytochrome oxidase containing 2 hemes a and one heme c, and a bc1 complex containing 2 hemes b and 2 hemes c. Gel electrophoresis in the presence of dodecyl sulfate showed that the immunoprecipitate and quinol oxidase preparation were composed of seven subunits; those of 51 (56-kDa), 38, and 22 kDa for cytochrome oxidase and those of 29, 23, 21, and 14 kDa for the bc1 complex. The 38-, 29-, and 21 kDa components possessed covalently bound heme c. The apparent molecular mass of the super complex was estimated to be as 380 kDa by gel filtration.
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PMID:Identification and properties of a quinol oxidase super-complex composed of a bc1 complex and cytochrome oxidase in the thermophilic bacterium PS3. 282 57

Membranes were isolated by French pressure cell extrusion of lysozyme-preincubated cells of the cyanobacterium Synechocystis 6714 after growth in the presence of 0.4 M NaCl for 4 days. These cells showed up to 6-fold respiratory activity (oxygen uptake) when compared to control cells. Separation of plasma and thylakoid membranes revealed that the major part of cytochrome c oxidase was associated with the latter. Immunoblotting of sodium dodecylsulfate polyacrylamide gel electrophorized membranes with antisera raised against subunit I, subunit II, and the holoenzyme of the aa3-type cytochrome oxidase from Paracoccus denitrificans gave specific and complementary cross-reactions at apparent molecular weights of about 25 and 17-18 kDa, respectively. Crude membranes were solubilized also with n-octyl glucoside, and the cytochrome oxidase was separated from the extract by affinity chromatography using immobilized cytochrome c from Saccharomyces cerevisiae. The enzyme was eluted with KCl/octyl glucoside. Dialysed and concentrated enzyme solution, which was free of b- and c-type cytochromes, gave reduced alpha- and gamma-peaks around 603 and 443 nm, respectively. Upon treatment of the sample with carbon monoxide the peaks were found at 593 and 433 nm, respectively. Photodissociation spectra of the CO-complexed enzyme were in full agreement with cytochrome aa3 being a functional cytochrome oxidase in Synechocystis 6714.
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PMID:Immunological and spectral characterization of partly purified cytochrome oxidase from the cyanobacterium Synechocystis 6714. 282 95

Octyl glucoside is an effective, nonionic, solubilizing agent for membrane proteins with the advantage of ease of removal by dialysis. In order to study the detergent-sensitive activity of cytochrome c oxidase, we chose this detergent because of its simple structure and the possibility of synthesizing analogues to test the structural dependence of the detergent specificity. A procedure was therefore developed that facilitates large-scale preparation of octyl glucoside and related alkyl glycosides, improving on previous methods by eliminating crystallization steps and employing a one-step purification of the final product on Dowex 1. This new purification procedure is particularly important for achieving the level of purity required to obtain the disaccharide, longer alkyl chain detergents in soluble form. Of the alkyl glycosides prepared (octyl beta-D-glucopyranoside, octyl beta-D-lactopyranoside, dodecyl beta-D-lactopyranoside, dodecyl beta-D-cellobiopyranoside, and dodecyl beta-D-maltopyranoside), lauryl (dodecyl) maltoside was found to be the most successful as an activator of purified beef and Neurospora cytochrome oxidase, giving two- to tenfold higher activities than octyl glucoside and other commercially available detergents, Tween-20 and Triton X-100. Kinetic studies using two different steady-state assay systems indicate that the activity changes are not the result of altered binding of the substrate but rather reflect a detergent effect on the state of association of the enzyme (as a monomer, dimer, or polymer) as well as on its intrinsic activity. By gel filtration procedures, lauryl maltoside and octyl glucoside were found to exist as monodisperse populations of micelles of 50 000 and 8000 daltons, respectively. The small uniform micelles and chemically well-defined structures of lauryl maltoside and octyl glucoside make them superior to other nonionic detergents for the study of membrane proteins in general and cytochrome oxidase in particular, since its activity in lauryl maltoside most closely approaches that of the physiological state.
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PMID:Alkyl glycoside detergents: a simpler synthesis and their effects on kinetic and physical properties of cytochrome c oxidase. 625 May 83

Most active transport across the bacterial cell membrane is driven by a proton electrochemical gradient (delta-muH+, interior negative and alkaline) generated via electron transfer through a membrane-bound respiratory chain. This phenomenon is now reproduced in vitro with proteoliposomes containing only two proteins purified from the membrane of Escherichia coli. An o-type cytochrome oxidase was extracted from membranes of a cytochrome d terminal oxidase mutant with octyl beta-D-glucopyranoside after sequential treatment with urea and cholate and was purified to homogeneity by ion-exchange chromatography. The purified oxidase contains four polypeptides (MrS 66,000, 35,000, 22,000, and 17,000), two b-type cytochromes (b558 and b563), and 16-17 nmol of heme b per mg of protein, and it catalyzes the oxidation of ubiquinol and other electron donors with specific activities 20- to 30-fold higher than crude membranes. The lac carrier protein was purified as described. Proteoliposomes were formed in the presence of the oxidase and lac carrier protein by detergent dilution, followed by freeze-thaw/sonication. The system generates a delta-muH+ (interior negative and alkaline) with ubiquinol as electron donor and the magnitude of delta-muH+ is dependent on the concentration of cytochrome o in the proteoliposomes. Furthermore, the proteoliposomes transport lactose against a concentration gradient to an extent that is commensurate with the magnitude of delta-muH+ generated. The results provide powerful additional support for the "chemiosmotic hypothesis" and demonstrate that purified lac carrier protein retains the ability to function in a physiological manner.
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PMID:Reconstitution of active transport in proteoliposomes containing cytochrome o oxidase and lac carrier protein purified from Escherichia coli. 630 57

A screen has been performed of possible inhibitors of the ubiquinol oxidase of higher plant mitochondria by assaying their effects on cyanide-insensitive NADH oxidase of mitochondria of Arum maculatum. A number of compounds which have powerful inhibitory effects have been identified. Potent inhibition was found with compounds related to the previously described n-propyl gallate, but with the n-propyl sidechain replaced with alkyl chains of greater hydrophobicity. Titration of a range of partial reactions showed that the inhibitors act specifically on the ubiquinol oxidase. The concentrations of inhibitor required are dependent on the respiratory substrate and on the amount of mitochondria used in the assay. Octyl gallate also proved to be a potent inhibitor of the ubiquinol oxidase in tobacco cell suspensions. A second class of compounds which strongly inhibit cyanide-insensitive NADH oxidation is aurachin C and its analogues. Compounds related to aurachin D are much less effective. Titrations of a range of partial reactions indicate that inhibition is caused by a direct action on the ubiquinol oxidase. However, both types of aurachins also act strongly at the Qi site of the cytochrome bc1 complex, as already known to be the case in other systems, and so they are of more limited value for studies of the ubiquinol oxidase. Titration of the oxidation of NADH via the ubiquinol oxidase in a purified mitochondrial fraction from the spadices of Arum maculatum with octyl gallate gave a half-maximal effect at a concentration of around 6 nM when the protein concentration was 14 micrograms ml-1. A similar titre was obtained with a decyl derivative of aurachin C. This allowed us to estimate an upper limit for the concentration of ubiquinol oxidase in these mitochondria of 0.72 +/- 0.15 nmol mg-1 protein, or a ratio of ubiquinol oxidase/cytochrome oxidase of about 15 +/- 7:1. The measurements also provide a minimal turnover number for the ubiquinol oxidase of 186 +/- 42 electrons.s-1. Titration of the ubiquinol oxidase in soybean cotyledon mitochondria with these compounds gave the concentration of inhibitor required to elicit 50% of the maximum observed effect (I50) values about one order of magnitude higher than those found with Arum mitochondria, and again the values depended on the respiratory substrate. An explanation for the variation in I50 values may be found in terms of differences in oxidase concentrations in the different mitochondrial membranes and in the differences in rate-controlling steps with substrates of different activities.
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PMID:New inhibitors of the ubiquinol oxidase of higher plant mitochondria. 758 98

The ba3-type cytochrome-c oxidase from Thermus thermophilus has been crystallized in its native form. Crystallization was achieved by the batch and the vapour diffusion sitting drop methods using polyethylene glycol monomethyl ether 2000 as precipitating agent in the presence of octyl-beta-D-thioglucoside as detergent. The crystals diffract to 3.8 A, belong to the space group P2 or P2(1) and have unit cell dimensions of a = 80.7 A; b = 116.0 A; c = 156.9 A and beta = 104.4 degrees. The asymmetric unit contains two ba3-type oxidase molecules.
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PMID:Three-dimensional crystals of cytochrome-c oxidase from Thermus thermophilus diffracting to 3.8 A resolution. 761 66

Complete DNA sequences encoding the Arabidopsis thaliana STP1 monosaccharide/H+ symporter or a histidine-tagged STP1-His6 protein were expressed in baker's yeast Saccharomyces cerevisiae. Both wild-type STP1 and the recombinant his-tagged protein were located in the plasma membranes of transformed yeast cells. The C-terminal modification caused no loss of transport activity compared with the wild-type protein. Anti-STP1-antibodies were used to confirm the identity of the protein in yeast and to compare the apparent molecular weights of STP1 proteins in membrane extracts from yeast or Arabidopsis thaliana. Purified yeast plasma membranes were fused with proteoliposomes consisting of Escherichia coli lipids and beef heart cytochrome-c oxidase. Addition of ascorbate/TMPD/cytochrome-c to these fused vesicles caused an immediate formation of membrane potential (inside negative; monitored with [3H]tetraphenylphosphonium cations) and a simultaneous, uncoupler-sensitive influx of D-glucose into the energized vesicles. STP1-His6 protein is functionally active after solubilization with octyl-beta-D-glucoside, which was shown by insertion of the protein into proteoliposomes by detergent dilution and determination of the resulting transport capacity. Detergent extracts from either total membranes or plasma membranes of transgenic yeast cells were used for one-step purification of the STP1-His6 protein on Ni(2+)-NTA columns. The identity of the purified protein was checked by immunoblotting and N-terminal sequencing.
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PMID:Functional reconstitution of the solubilized Arabidopsis thaliana STP1 monosaccharide-H+ symporter in lipid vesicles and purification of the histidine tagged protein from transgenic Saccharomyces cerevisiae. 792 Jul 12

Inner membranes were prepared from Escherichia coli strain RG 145, which is deficient in cytochrome bd, but overexpresses cytochrome bo [Au and Gennis (1987) J. Bacteriol. 169, 3237-3242]. The latter was purified 7-fold by extracting the membranes with octyl beta-D-glucopyranoside, followed by chromatography on DEAE-Sepharose, yielding 150 mg of protein/150 g wet weight of cells. Optical e.p.r. and low-temperature m.c.d. (magnetic circular dichroism) spectroscopies were used to investigate the nature of the protein ligands to the two haems in cytochrome bo from E. coli. Low-spin ferric haem b, the origin of a rhombic e.p.r. spectrum with g = 2.98, 2.26 and 1.50, gives rise to a charge-transfer band in the near-i.r. m.c.d. spectrum at 1622 nm. It is therefore concluded that haem b is co-ordinated by two histidine residues. The low-temperature m.c.d. spectrum of dithionite-reduced cytochrome bo comprises bands due both to low-spin ferrous haem b and to high-spin ferrous haem o. The bands arising from haem o show a direct correspondence with those in the m.c.d. spectrum of five-co-ordinate histidine-ligated ferrous haems such as myoglobin, implying that the protein residue liganding haem o is also histidine. This assignment was confirmed by measuring the e.p.r. spectrum of the nitric oxide derivative of fully reduced cytochrome bo. This showed a rhombic spectrum with g = 2.098, 2.008 and 1.987, and nuclear hyperfine splitting consistent with the co-ordination of ferrous haem by NO and histidine. The hyperfine splittings observed were 1.95 +/- 0.05 mT for the 14N of the NO ligand and 0.75 +/- 0.05 mT for the 14N of the proximal histidine. The e.p.r. spectrum of some samples of oxidized cytochrome bo show, at temperatures below 15 K, broad signals at g = 7.6, 3.6 and 2.8, and other preparations in the presence of glycerol yield signals at g = 10.8, 3.2 and 2.6. These signals, which are abolished by the addition of cyanide, are assigned to the binuclear centre, cytochrome o-CuB, suggesting that the binuclear site may display heterogeneity. Carbon monoxide reacts with the reduced enzyme with a stoichiometry of 1:1, and the dissociation constant for this reaction was determined to be 1.7 x 10(-6)M. The second-order rate constants for this reaction were measured and shown to be similar to those determined for bovine cytochrome aa3 [Gibson and Greenwood (1963) Biochem. J. 86, 541-554].
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PMID:Cytochrome bo from Escherichia coli: identification of haem ligands and reaction of the reduced enzyme with carbon monoxide. 838 47

A prokaryotic biotin acceptor domain was fused to the carboxy terminal end of the Chlorella hexose-proton symporter. The plant symporter is biotinylated in vivo when expressed in Schizosaccharomyces pombe. The extended biotinylated transport protein is fully active, catalyzes accumulation of D-glucose analogs and restores growth of a glucose-uptake-deficient yeast strain. Crude membranes were solubilized with octyl-beta-D-glucoside in the presence of Escherichia coli L-alpha-phosphatidylethanolamine. Biotinylated symporter was purified to homogeneity by biotinavidin affinity chromatography. The symporter protein was reconstituted together with cytochrome-c oxidase prepared from beef heart mitochondria into proteo-liposomes. Cytochrome-c oxidase is a redox-driven H(+)-pump generating a proton motive force (inside negative and alkaline) while transferring electrons from cytochrome-c to oxygen; this energy is used by the symporter to accumulate D-glucose at least 30-fold. In the absence of the driving force the transport protein facilitates diffusion of D-glucose until the concentration equilibrium is reached. It was shown that maximal transport activity depends highly on the amount of co-reconstituted cytochrome-c oxidase and that the symporter possesses 10% of its in vivo turnover number under optimized in vitro transport conditions.
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PMID:Purification of the Chlorella HUP1 hexose-proton symporter to homogeneity and its reconstitution in vitro. 901 Oct 86

Rhizopus stolonifer (Ehrenb.:Fr.) Vuill mitochondria contain the complete system for oxidative phosphorylation, formed by the classical components of the electron transport chain (complexes I, II, III, and IV) and the F(1)F(0)-ATP synthase (complex V). Using the native gel electrophoresis, we have shown the existence of supramolecular associations of the respiratory complexes. The composition and stoichiometry of the oxidative phosphorylation complexes were similar to those found in other organisms. Additionally, two alternative routes for the oxidation of cytosolic NADH were identified: the alternative NADH dehydrogenase and the glycerol-3-phosphate shuttles. Residual respiratory activity after inhibition of complex IV by cyanide was inhibited by low concentrations of n-octyl gallate, indicating the presence of an alternative oxidase. The K(0.5) for the respiratory substrates NADH, succinate, and glycerol-3-phosphate in permeabilized cells was higher than in isolated mitochondria, suggesting that interactions of mitochondria with other cellular elements might be important for the function of this organelle.
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PMID:The mitochondrial respiratory chain of Rhizopus stolonifer (Ehrenb.:Fr.) Vuill. 2306 42


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