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Query: UNIPROT:O95477 (membrane-bound)
 29,236 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Escherichia coli grew anaerobically on a minimal medium with glycerol as the carbon and energy source and dimethyl sulfoxide (DMSO) as the terminal electron acceptor. DMSO reductase activity, measured with an artificial electron donor (reduced benzyl viologen), was preferentially associated with the membrane fraction (77 +/- 10% total cellular activity). A Km for DMSO reduction of 170 +/- 60 microM was determined for the membrane-bound activity. Methyl viologen, reduced flavin mononucleotide, and reduced flavin adenine dinucleotide also served as electron donors for DMSO reduction. Methionine sulfoxide, a DMSO analog, could substitute for DMSO in both the growth medium and in the benzyl viologen assay. DMSO reductase activity was present in cells grown anaerobically on DMSO but was repressed by the presence of nitrate or by aerobic growth. Anaerobic growth on DMSO coinduced nitrate, fumarate, and and trimethylamine-N-oxide reductase activities. The requirement of a molybdenum cofactor for DMSO reduction was suggested by the inhibition of growth and a 60% reduction in DMSO reductase activity in the presence of 10 mM sodium tungstate. Furthermore, chlorate-resistant mutants chlA, chlB, chlE, and chlG were unable to grow anaerobically on DMSO. DMSO reduction appears to be under the control of the fnr gene.
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PMID:Dimethyl sulfoxide reductase activity by anaerobically grown Escherichia coli HB101. 388 58

Trimethylamine oxide, which is found in relatively high concentrations in the tissues of marine animals, serves as an electron acceptor in the anaerobic metabolism of a number of bacteria associated primarily with three environments: the marine environment (e.g. Alteromonas and Vibrio), the brackish pond (nonsulfur photosynthetic bacteria), and animal intestines (Enterobacteriaceae). Its reduction to trimethylamine by such bacteria can constitute a major spoilage reaction during the storage of marine fish. In the Enterobacteriaceae, anaerobic respiration with TMAO has been shown to support oxidative phosphorylation. Electron transport to TMAO in these bacteria involves flavin nucleotides, menaquinones, both b- and c-type cytochromes, and a molybdoenzyme reductase. Formate, hydrogen, lactate, and glycerol all serve as electron donors for TMAO respiration. Electrophoretically distinct constitutive and TMAO-induced reductases are synthesized by both E. coli and S. typhimurium. Electron transport to TMAO is repressed both by air and by nitrate. A number of genes involved in TMAO respiration have been mapped, but the structural gene for the inducible TMAO reductase has not yet been firmly established. Oxidative phosphorylation is also supported by TMAO reduction in Alteromonas. In this organism, which is nonfermentative, TMAO respiration resembles aerobic respiration in that intermediates of the TCA cycle are excellent electron donors. Alteromonas exhibits a requirement for NaCl for growth on TMAO and certain electron donors. As in the Enterobacteriaceae, air and nitrate both interfere with TMAO reduction. The role of TMAO reduction in the anaerobic metabolism of nonsulfur purple bacteria has not yet been resolved; it is not clear if TMAO serves simply as an accessory oxidant for fermentation or if TMAO reduction is associated with energy-yielding membrane-bound electron transport. Some of the confusion regarding this bacterial group stems from the fact that much of the work to date has involved parallel studies of TMAO and dimethyl sulfoxide reduction, and it is not yet known whether the two compounds are reduced by the same enzyme. Although our understanding of bacterial TMAO reduction lags far behind our knowledge of bacterial nitrate reduction, it is unlikely that this will always be the case.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Bacterial reduction of trimethylamine oxide. 390 97

The coupling of membrane-bound glucose dehydrogenase (EC 1.1.99.17) to the respiratory chain has been studied in whole cells, cell-free extracts, and membrane vesicles of gram-negative bacteria. Several Escherichia coli strains synthesized glucose dehydrogenase apoenzyme which could be activated by the prosthetic group pyrrolo-quinoline quinone. The synthesis of the glucose dehydrogenase apoenzyme was independent of the presence of glucose in the growth medium. Membrane vesicles of E. coli, grown on glucose or succinate, oxidized glucose to gluconate in the presence of pyrrolo-quinoline quinone. This oxidation led to the generation of a proton motive force which supplied the driving force for uptake of lactose, alanine, and glutamate. Reconstitution of glucose dehydrogenase with limiting amounts of pyrrolo-quinoline quinone allowed manipulation of the rate of electron transfer in membrane vesicles and whole cells. At saturating levels of pyrrolo-quinoline quinone, glucose was the most effective electron donor in E. coli, and glucose oxidation supported secondary transport at even higher rates than oxidation of reduced phenazine methosulfate. Apoenzyme of pyrrolo-quinoline quinone-dependent glucose dehydrogenases with similar properties as the E. coli enzyme were found in Acinetobacter calcoaceticus (var. lwoffi) grown aerobically on acetate and in Pseudomonas aeruginosa grown anaerobically on glucose and nitrate.
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PMID:Energy transduction by electron transfer via a pyrrolo-quinoline quinone-dependent glucose dehydrogenase in Escherichia coli, Pseudomonas aeruginosa, and Acinetobacter calcoaceticus (var. lwoffi). 392 46

The composition of the membrane-bound electron transport system of Haemophilus parainfluenzae underwent modification in response to the terminal electron acceptor in the growth medium. H. parainfluenzae was able to grow with O(2), nitrate, fumarate, pyruvate, and substrate amounts of nicotinamide adenine dinucleotide (NAD) as electron acceptors. When O(2) served as the electron acceptor and its concentration was lowered below 20 mum, the bacteria formed more cytochromes b, c, a(1), a(2), and o than were present in the cells grown at 150 to 200 mum O(2). Nitrate and nitrite reductase activities also appeared during growth at the low O(2) concentrations in the absence of added nitrate. Cytochrome levels in cells grown anaerobically with fumarate, pyruvate, or NAD as terminal acceptors were similar to those formed in cells grown at low O(2) concentrations. Cells grown with nitrate had higher levels of cytochromes c, b, and o, and of nitrate and nitrite reductases, than did cells grown with the other acceptors. The formation of cytochrome oxidase a(2) was repressed by the presence of nitrate in the growth medium. The critical O(2) concentration (the O(2) concentration at which the rate of O(2) uptake becomes demonstrably dependent on the O(2) concentration) was about 100 mum in cells grown with nitrate and about 15 mum in cells grown with the other acceptors. A mutant of H. parainfluenzae was found to make about 10% as much cytochrome c as the wild type, and its formation of cytochrome a(2) was not repressed by nitrate. The critical O(2) concentration of the mutant was high when it was grown with nitrate, suggesting that the high levels of cytochrome c and the absence of cytochrome a(2) from the wild type are not responsible for the high critical O(2) concentration. The modifications of the respiratory system induced by changing the terminal electron acceptor were inhibited by the presence of chloramphenicol, which suggests that protein synthesis is involved.
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PMID:Effect of nitrate, fumarate, and oxygen on the formation of the membrane-bound electron transport system of Haemophilus parainfluenzae. 431 51

White bands resulting from precipitation of dodecan-1-ol liberated by hydrolysis of sodium dodecyl sulfate and decan-5-ol released by hydrolysis of decan-5-yl sulfate produced zymograms of the primary and secondary alkylsulfatases from Pseudomonas C(12)B. Gas-liquid chromatographic analyses of ether extracts of the precipitate-containing segments of the zymograms confirmed the identity of the alcohols which were not discerned in extracts of segments of the gels other than those containing precipitates. beta-Galactosidase from Escherichia coli was marked on zymograms by the liberation of o-nitrophenol from o-nitrophenyl-beta-D-galactoside, and arylsulfatase from Pseudomonas C(12)B was marked in gels by liberation of p-nitrophenol from p-nitrophenyl sulfate. Membrane-associated dissimilatory nitrate reductases from a nitrate respirer (Enterobacter aerogenes) and a denitrifier (Pseudomonas perfectomarinus) did not penetrate either 6.8 or 3% polyacrylamide gel but were demonstrable at the top of the gels. In the membrane-bound state, formate served as electron donor for nitrate reductase from E. aerogenes, and reduced nicotinamide adenine dinucleotide (NADH) served as donor for nitrate reductase from P. perfectomarinus. Both enzymes reduced nitrate at the expense of reduced benzyl viologen as well. Assimilatory nitrate reductase from E. aerogenes moved easily into the 6.8% gels (R(f) = 0.43 under the conditions of these experiments). The reduced dye served as electron donor for the assimilatory reductase, but formate and NADH did not. Incubation of the membrane-associated nitrate reductases with 2% Triton X-100 solubilized the enzymes and removed the capacity of formate and NADH to serve as electron donors. Both retained the ability to reduce nitrate at the expense of reduced benzyl viologen. The solubilized dissimilatory reductase from E. aerogenes moved further in the gels (R(f) = 0.49) than the soluble assimilatory reductase; the solubilized dissimilatory reductase from the denitrifier, P. perfectomarinus, moved further in the gels (R(f) = 0.64) than either of the enzymes from E. aerogenes.
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PMID:Methods for visualization of enzymes in polyacrylamide gels. 435 59

The use of bacterial membrane vesicles as an experimental system for the study of active transport has been discussed. Vesicles are prepared from osmotically sensitized bacteria, and consist of osmotically intact, membranebound sacs without internal structure. They retain litle or no cytoplasm. Under appropriate conditions, these vesicles catalyze the transport of a variety of solutes at rates which are comparable, in many cases, to those of intact cells. Two general types of transport systems have been elucidated in the vesicle system: (i) group translocation systems which catalyze vectorial covalent reactions; and (ii) respirationlinked transport systems that catalyze the active transport of a whole range of metabolites against an electrochemical or osmotic gradient. In E. coli membrane vesicles, the respiration-linked transport systems are coupled primarily to the oxidation of (D)-lactate to pyruvate, catalyzed by a flavin-linked, membrane-bound (D)-lactate dehydrogenase which has been purified to homogeneity. Electrons derived from (D)-lactate or certain artificial electron donors are transferred to oxygen by means of a membrane-bound respiratory chain, and respiration is coupled to active transport within a segment of the respiratory chain between the primary dehydrogenase and cytochrome. b(l). The great majority of the individual membrane vesicles in the population catalyze active transport, and the generation or hydtolysis of ATP is not involved. Under anaerobic conditions, fumarate or nitrate can be utilized in place of oxygen as terminal electron acceptors. With the exception that (D)-lactate is not always the most effective electron donor for active transport, vesicles prepared from a number of other organisms catalyze transport in a similar manner. Fluorescent dansylgalactosides are useful molecular probes of active transport in the vesicle system. These compounds are competitive inhibitors of beta-galactoside transport, but are not transported themselves. Fluorescence studies indicate that the lac carrier protein constitutes approximately 3 to 6 percent of the total membrane protein, and that it is not accessible to the external medium unless the membrane is "energized." Thus, energy is coupled to one of the initial steps in the transport process. Studies with a photoaffinity-labeled galactoside provide independent support for this conclusion. When membrane vesicles prepared from a (D)-lactate dehydrogenase mutant of E. coli are treated with (D)-lactate dehydrogenase, the enzyme binds to the vesicles and they regain the capacity to catalyze (D)-lactate oxidation and (D)-lactate-dependent active transport. The maximal specific transport activity obtained in the reconstituted system is similar in magnitude to that of wildtype vesicles. Titration studies with dansylgalactoside demonstrate that there is at least a seven- to eightfold excess of lac carrier protein relative to (D)-lactate dehydrogenase. Evidence is presented indicating that the enzyme is bound to the inner surface of native membrane vesicles and to the outer surface of reconstituted vesicles, and that the flavin coenzyme moiety is critically involved in binding. Possible mechanisms of respirationlinked active transport are discussed.
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PMID:Transport studies in bacterial membrane vesicles. 462 43

Chlorate-resistant mutants corresponding to each known genetic locus (chlA, chlB, chlC, chlD, chlE) were isolated from Escherichia coli K-12. All these mutants showed decreased amounts of membrane-bound nitrate reductase, cytochrome b, and formic dehydrogenase, but all had normal succinic dehydrogenase activity. Proteins from the cytoplasmic membranes of these mutants were compared to those of the wild type-on polyacrylamide gels. The addition of nitrate to wild-type anaerobic cultures caused increased formation of three membrane proteins. These same proteins, along with one other, were missing in varying patterns in mutants altered at the different genetic loci. One of the missing proteins was found to be the enzyme nitrate reductase, although this protein was present in some mutants lacking nitrate reductase activity. None of the others has been identified.
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PMID:Alterations in the cytoplasmic membrane proteins of various chlorate-resistant mutants of Escherichia coli. 494 70

ChlD mutants of Escherichia coli are pleiotropic, lacking formate-nitrate reductase activity as well as formate-hydrogenlyase activity. Whole-chain formate-nitrate reductase activity, assayed with formate as the electron donor and measuring the amount of nitrite produced, was restored to wild-type levels in the mutants by addition of 10(-4)m molybdate to the growth medium. Under these conditions, the activity of each of the components of the membrane-bound nitrate reductase chain increased after molybdate supplementation. In the absence of nitrate, the activities of the formate-hydrogenlyase system were also restored by molybdate. Strains deleted for the chlD gene responded in a similar way to molybdate supplementation. The concentration of molybdenum in the chlD mutant cells did not differ significantly from that in the wild-type cells at either low or high concentrations of molybdate in the medium. However, the distribution of molybdenum between the soluble protein and membrane fractions differed significantly from wild type. We conclude that the chlD gene product cannot be a structural component of the formate-hydrogenlyase pathway or the formate-nitrate reductase pathway, but that it must have an indirect role in processing molybdate to a form necessary for both electron transport systems.
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PMID:Phenotypic restoration by molybdate of nitrate reductase activity in chlD mutants of Escherichia coli. 494 67

Two isozymes of aspartate aminotransferase have been demonstrated biochemically. One isozyme is found in the mitochondrial fraction of the cytoplasm, the other ("soluble") in the supernatant. Both isozymes can be demonstrated by the cytochemical technique of Lee and Torack, as reported in the preceding report. Aldehyde fixation rapidly inactivates both isozymes, especially the soluble one. Inactivation can be delayed by addition of ketoglutarate to the fixative. The ketoglutarate probably competes with the fixative for the active site of the enzyme, thus protecting that region of the molecule. This enables adequate tissue preservation with enough remaining enzymatic activity to be demonstrated by the precipitation of oxaloacetate as the lead salt from a medium containing alpha-ketoglutaric acid aspartic acid, and lead nitrate. Electron-opaque material was found not only in mitochondria but, as the result of substrate protection, on the plasma membranes of many cells including erythrocytes and bacteria, the limiting membrane of peroxisomes, and the transverse tubular system of striated muscle. Occasional centrioles, neurotubules, tubules in the tails of spermatozoa, the A-I band junction in myofibrils of striated muscle, and the ground substance between cisternae of endoplasmic reticulum in intestinal goblet cells also showed precipitate. In all cases, replacement of L-aspartic acid by D-aspartic acid in the medium resulted in unstained sections. The sensitivity of extramitochondrial sites to fixation, the need of ketoglutarate as an agent for protecting the enzymatic activity during the fixation process, and the known presence of only soluble isozyme in erythrocytes indicate that enzymatic activity at these sites can be attributed to the soluble isozyme. Localization of the soluble isozyme on the plasma membrane may be related to possible involvement in depolarization phenomena, amino acid transport, or synthesis of plasma membrane-bound mucopolysaccharides.
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PMID:The ultrastructural localization of the isozymes of aspartate aminotransferase in murine tissues. 553 35

Crossed immunoelectrophoresis was used to analyze the components of membrane vesicles of anaerobically grown Escherichia coli. The number of precipitation lines in the crossed immunoelectrophoresis patterns of membrane vesicles isolated from E. coli grown anaerobically on glucose plus nitrate and on glycerol plus fumarate were 83 and 70, respectively. Zymogram staining techniques were used to identify immunoprecipitates corresponding to nitrate reductase, formate dehydrogenase, fumarate reductase, and glycerol-3-phosphate dehydrogenase in crossed immunoelectrophoresis reference patterns. The identification of fumarate reductase by its succinate oxidizing activity was confirmed with purified enzyme and with mutants lacking or overproducing this enzyme. In addition, precipitation lines were found for hydrogenase, cytochrome oxidase, the membrane-bound ATPase, and the dehydrogenases for succinate, malate, dihydroorotate, D-lactate, 6-phosphogluconate, and NADH. Adsorption experiments with intact and solubilized membrane vesicles showed that fumarate reductase, hydrogenase, glycerol-3-phosphate dehydrogenase, nitrate reductase, and ATPase are located at the inner surface of the cytoplasmic membrane; on the other hand, the results suggest that formate dehydrogenase is a transmembrane protein.
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PMID:Identification and localization of enzymes of the fumarate reductase and nitrate respiration systems of escherichia coli by crossed immunoelectrophoresis. 621 54


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