Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0272170 (SDS)
 50,377 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Nucleotide pyrophosphatase was purified from human placenta to near homogeneity with a specific activity of about 500-fold over the Triton extract of the homogenate. Purification was achieved most effectively by successive chromatographic steps with AMP-agarose and ADP-agarose columns, based on the affinity of the enzyme towards 5'-adenylate and adenosine 3',5'-diphosphate, and a lectin-Sepharose column, based on the glycoprotein nature of the enzyme. The purified enzyme was found to be essentially homogeneous on SDS-polyacrylamide gel electrophoresis with a mobility corresponding to 130K. The purified enzyme was found to hydrolyze a wide variety of nucleotides, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), adenosine 5'-phosphosulfate (APS), NADH, ATP, nucleotide sugars, oligonucleotides, and p-nitrophenyl-thymidine 5'-phosphate (PNTP). From the oligonucleotides, the enzyme produced 5'-phosphates. Mg2+ was required for full activity. Glycine and sulfhydryl compounds such as 2-mercaptoethanol and 2,3-dimercapto-1-propanol were inhibitory. Most of these properties are common to nucleotide pyrophosphatases [EC 3.6.1.9] and type I (5'-phosphate forming) phosphodiesterases [EC 3.1.4.1] from various sources. The relevance of this enzyme to a unique genetic disease, Lowe's syndrome, is discussed.
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PMID:Purification and properties of nucleotide pyrophosphatase from human placenta. 300 Oct 38

A homogeneous multimeric protein isolated from the green alga, Scenedesmus obliquus, has both latent phosphoribulokinase activity and glyceraldehyde-3-phosphate dehydrogenase activity. The glyceraldehyde-3-phosphate dehydrogenase was active with both NADPH and NADH, but predominantly with NADH. Incubation with 20 mM dithiothreitol and 1 mM NADPH promoted the coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase, accompanied by a decrease in the glyceraldehyde-3-phosphate dehydrogenase activity linked to NADH. The multimeric enzyme had a Mr of 560,000 and was of apparent subunit composition 8G6R. R represents a subunit of Mr 42,000 conferring phosphoribulokinase activity and G a subunit of 39,000 responsible for the glyceraldehyde-3-phosphate dehydrogenase activity. On SDS-PAGE the Mr-42,000 subunit comigrates with the subunit of the active form of phosphoribulokinase whereas that of Mr-39,000 corresponds to that of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. The multimeric enzyme had a S20,W of 14.2 S. Following activation with dithiothreitol and NADPH, sedimenting boundaries of 7.4 S and 4.4 S were formed due to the depolymerization of the multimeric protein to NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (4G) and active phosphoribulokinase (2R). It has been possible to isolate these two enzymes from the activated preparation by DEAE-cellulose chromatography. Prolonged activation of the multimeric protein by dithiothreitol in the absence of nucleotide produced a single sedimenting boundary of 4.6 S, representing a mixture of the active form of phosphoribulokinase and an inactive dimeric form of glyceraldehyde-3-phosphate dehydrogenase. Algal thioredoxin, in the presence of 1 mM dithiothreitol and 1 mM NADPH, stimulated the depolymerization of the multimeric protein with resulting coactivation of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase. Light-induced depolymerization of the multimeric protein, mediated by reduced thioredoxin, is postulated as the mechanism of light activation in vivo. Consistent with such a postulate is the presence of high concentrations of the active forms of phosphoribulokinase and NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase in extracts from photoheterotrophically grown algae. By contrast, in extracts from the dark-grown algae the multimeric enzyme predominates.
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PMID:Properties of a multimeric protein complex from chloroplasts possessing potential activities of NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase and phosphoribulokinase. 302 12

NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.
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PMID:Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction. 309 Oct 6

A mitochondrial NADH:Q6 oxidoreductase has been isolated from cells of Saccharomyces cerevisiae by a simple method involving extraction of the enzyme from the mitochondrial membrane with Triton X-100, followed by chromatography on DEAE-cellulose and blue Sepharose CL-6B. By this procedure a 2000-fold purification is achieved with respect to whole cells or a 150-fold purification with respect to the mitochondrion. The purified NADH dehydrogenase consists of a single subunit with molecular mass of 53 kDa as indicated by SDS/polyacrylamide gel electrophoresis. The enzyme contains FAD, non-covalently linked, as the sole prosthetic group with Em,7.6 = -370 mV and no iron-sulphur clusters. The enzyme is specific for NADH with apparent Km = 31 microM and was found to be inhibited by flavone (I50 = 95 microM), but not by rotenone or piericidin. The purified enzyme can use ubiquinone-2, -6 or -10, menaquinone, dichloroindophenol or ferricyanide as electron acceptors, but at different rates. The greatest turnover of NADH was obtained with ubiquinone-2 as acceptor (2500 s-1). With the natural ubiquinone-6 this value was 500 s-1. The NADH:Q2 oxidoreductase activity shows a maximum at pH 6.2, the NADH:Q6 oxidoreductase activity is constant between pH 4.5-9.0. The amount of enzyme in the cell is subject to glucose repression; it increases slightly when cells, grown on glucose or lactate, enter the stationary phase. The experiments performed so far suggest that the enzyme purified in this study is the external NADH:Q6 oxidoreductase, bound to the mitochondrial inner membrane and that it is involved in the oxidation of cytosolic NADH. The relation of this enzyme with respect to various other NADH dehydrogenases from yeast and plant mitochondria is discussed.
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PMID:Purification and characterization of a rotenone-insensitive NADH:Q6 oxidoreductase from mitochondria of Saccharomyces cerevisiae. 313 18

The present paper describes the analysis of plant mitochondrial NADH dehydrogenases using a recently developed non-dissociating gradient polyacrylamide-gel-electrophoresis technique [Kuonen, Roberts & Cottingham (1986) Anal. Biochem. 153, 221-226]. Solubilized mung-bean (Phaseolus aureus) submitochondrial particles were analysed on 3-22% (w/v) gradient polyacrylamide gels containing 0.1% Triton X-100 and stained for multiple NADH dehydrogenase activities. A rotenone-sensitive NADH dehydrogenase (Complex I) was identified on the basis of co-migration with the purified mammalian enzyme. The polypeptide composition of the plant enzyme was further analysed by band excision and SDS/polyacrylamide-gel electrophoresis.
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PMID:Analysis of NADH dehydrogenases from plant [mung bean (Phaseolus aureus)] mitochondrial membranes on non-denaturing polyacrylamide gels and purification of complex I by band excision. 317 53

Walker tumour cells in vivo or in vitro are exceptionally sensitive to the monofunctional alkylating agent 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) (Cobb LM et al., Biochem Pharmacol 18: 1519-1527, 1969). CB 1954 forms DNA interstrand crosslinks in a time-dependent manner in Walker tumour cells but not in non-toxically affected Chinese hamster V79 cells [(Roberts JJ et al., Biochem Biophys Res Commun 140: 1073-1078, 1986)]. However, co-culturing Chinese hamster V79 cells with Walker cells in the presence of CB 1954 renders the hamster cells sensitive to CB 1954 and leads to the formation of interstrand crosslinks in their DNA, findings indicative of the formation by Walker cells of a diffusible toxic metabolite of CB 1954. A flavoprotein, of molecular weight 33.5 kDa as estimated by SDS-polyacrylamide gel electrophoresis, has been isolated from Walker cells and identified as a form of NAD(P)H dehydrogenase (quinone) (DT diaphorase, EC 1.6.99.2). This enzyme, in the presence of NADH or NADPH, catalyses the aerobic reduction of CB 1954 to 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide. This new compound can form interstrand crosslinks in the DNA of Chinese hamster V79 cells to which it is also highly toxic.
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PMID:A new cytotoxic, DNA interstrand crosslinking agent, 5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide, is formed from 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB 1954) by a nitroreductase enzyme in Walker carcinoma cells. 320 2

The NAD-dependent glutamate dehydrogenase from Phycomyces spores was purified more than 300-fold. Estimation of Mr by gel filtration gave a value of 98,000 whereas after SDS-PAGE one major band of Mr 54,000 was found, suggesting that the enzyme is a dimer. The enzyme was virtually dependent on the presence of AMP for activity and showed half-maximal activation at 9.5 and 43 microM-AMP in the direction of animation and deamination respectively. ADP was nearly as effective at 20-fold higher concentrations. Other nucleotide monophosphates were ineffective and nucleoside triphosphates were slightly inhibitory. Hyperbolic kinetics were found for all substrates yielding Km values of about 10 mM for ammonium, 1 mM for 2-oxoglutarate and 0.1 mM for NADH in the direction of amination, and 10 mM for glutamate and 0.7 mM for NAD in the direction of deamination.
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PMID:Purification and properties of NAD-dependent glutamate dehydrogenase from Phycomyces spores. 322 Dec

A new enzyme, N-acyl-D-mannosamine dehydrogenase, was purified to apparent homogeneity from a cell-free extract of Flavobacterium sp. 141-8 and some of its properties were investigated. The enzyme showed optimum activity at pH 8.0-9.5. N-Acetyl- and N-glycolyl-D-mannosamine were oxidized but other commonly existing sugars, such as N-acetylglucosamine, N-acetylgalactosamine, amino sugars, neutral hexoses, and pentoses, were not oxidized. NAD+ was specifically utilized as an effective hydrogen acceptor. The apparent Km values for N-acetyl- and N-glycolyl-D-mannosamine, and NAD+ were 1.0, 13.3, and 0.41 mM, respectively. The stoichiometry data showed that 1 mol each of N-acetyl-D-mannosamine and NAD+ were converted to 1 mol each of N-acetyl-D-mannosaminic acid and NADH, respectively. Although the formation of lactone was detected in the enzyme reaction mixture, the reverse reaction of the enzyme, the reduction of N-acetyl-D-mannosamino-lactone, was not observed. The enzyme activity was strongly inhibited by Hg2+ and SDS, but metal-chelating reagents and sulfhydryl-group-blocking reagents had almost no effect. The molecular weight of the enzyme was estimated to be 120,000 on gel filtration and 29,000 on SDS-polyacrylamide gel electrophoresis. Its isoelectric point was at pH 4.8. On trial application of the enzyme, it was indicated that N-acetylneuraminic acid can be determined quantitatively with the combined enzyme system involving the new enzyme and N-acetylneuraminic acid aldolase.
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PMID:Purification and properties of N-acyl-D-mannosamine dehydrogenase from Flavobacterium sp. 141-8. 324 Sep 88

Asp-362, a potential key catalytic residue of Escherichia coli citrate synthase (citrate oxaloacetate-lyase [pro-3S)-CH2COO- ----acetyl-CoA), EC 4.1.3.7) has been converted to Gly-362 by oligonucleotide-directed mutagenesis. The mutant gene was completely sequenced, using a series of synthetic oligodeoxynucleotides spanning the structural gene to confirm that no additional mutations had occurred during genetic manipulation. The mutant gene was expressed in M13 bacteriophage and produced a protein which migrated in an identical manner to wild-type E. coli citrate synthase on SDS-polyacrylamide gels and which cross-reacted with E. coli citrate synthase antiserum. The mutant gene was subsequently recloned into pBR322 for large scale purification of the protein, and the resulting plasmid, pCS31, used to transform the citrate synthase deletion strain, W620. The mutant enzyme purified in an analogous manner to wild-type E. coli citrate synthase and expressed less than 2% of wild-type enzyme activity. The activity of the partial reactions catalysed by citrate synthase was similarly affected suggesting that this residual activity may be due to contaminating wild-type enzyme activity. The mutant citrate synthase retains a high-affinity NADH-binding site consistent with the protein preserving its overall structural integrity. Oxaloacetate binding to the protein is unaffected by the Asp-362 to Gly-362 mutation. Binding of the acetyl-CoA analogue, carboxymethyl-CoA, could not be detected in the mutant protein indicating that the lack of catalytic competence is due primarily to the inability of the protein to bind the second substrate, acetyl-CoA.
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PMID:Site-directed mutagenesis of citrate synthase; the role of the active-site aspartate in the binding of acetyl-CoA but not oxaloacetate. 328 13

A quick, reliable, purification procedure was developed for purifying both benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from a single batch of Acinetobacter calcoaceticus N.C.I.B. 8250. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernatant, followed by chromatography on DEAE-Sephacel, affinity chromatography on Blue Sepharose CL-6B and Matrex Gel Red A, and finally gel filtration through a Superose 12 fast-protein-liquid-chromatography column. The enzymes co-purified as far as the Blue Sepharose CL-6B step were separated on the Matrex Gel Red A column. The final preparations of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II gave single bands on electrophoresis under non-denaturing conditions or on SDS/polyacrylamide-gel electrophoresis. The enzymes are tetramers, as judged by comparison of their subunit (benzyl alcohol dehydrogenase, 39,700; benzaldehyde dehydrogenase II, 55,000) and native (benzyl alcohol dehydrogenase, 155,000; benzaldehyde dehydrogenase II, 222,500) Mr values, estimated by SDS/polyacrylamide-gel electrophoresis and gel filtration respectively. The optimum pH values for the oxidation reactions were 9.2 for benzyl alcohol dehydrogenase and 9.5 for benzaldehyde dehydrogenase II. The pH optimum for the reduction reaction for benzyl alcohol dehydrogenase was 8.9. The equilibrium constant for oxidation of benzyl alcohol to benzaldehyde by benzyl alcohol dehydrogenase was determined to be 3.08 x 10(-11) M; the ready reversibility of the reaction catalysed by benzyl alcohol dehydrogenase necessitated the development of an assay procedure in which hydrazine was used to trap the benzaldehyde formed by the NAD+-dependent oxidation of benzyl alcohol. The oxidation reaction catalysed by benzaldehyde dehydrogenase II was essentially irreversible. The maximum velocities for the oxidation reactions catalysed by benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II were 231 and 76 mumol/min per mg of protein respectively; the maximum velocity of the reduction reaction of benzyl alcohol dehydrogenase was 366 mumol/min per mg of protein. The pI values were 5.0 for benzyl alcohol dehydrogenase and 4.6 for benzaldehyde dehydrogenase II. Neither enzyme activity was affected when assayed in the presence of a range of salts. Absorption spectra of the two enzymes showed no evidence that they contain any cofactors such as cytochrome, flavin, or pyrroloquinoline quinone. The kinetic coefficients of the purified enzymes with benzyl alcohol, benzaldehyde, NAD+ and NADH are also presented.
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PMID:Benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase II from Acinetobacter calcoaceticus. Purification and preliminary characterization. 329 54


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