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)

The NAD-dependent glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) of the salt-tolerant yeast Debaryomyces hansenii was purified by poly(ethylene glycol) precipitation and a combination of chromatographic procedures. The enzyme existed in two forms with different ionic characters and specific activity. On SDS-polyacrylamide gel electrophoresis, both forms yielded one predominant band with an apparent molecular weight of 42,000. The specific activity of the enzyme was dependent on the concentration of the enzyme and on the ionic strength of the dissolving medium. All ions tested stimulated the enzyme activity in the ionic strength range 0-100 mM, with glutamate yielding the highest activity. Above these concentrations, the dehydrogenase showed high tolerance for glutamate in concentrations up to 0.9 M, whereas malate, sulfate and chloride were inhibitory. Enzyme activity showed little sensitivity to the type of cation present and was only slightly affected by 5 M glycerol. The true Km values for the substrates were 6.6 microM for NADH, 130 microM for dihydroxyacetone phosphate, 0.3 mM for NAD and 1.2 mM for glycerol-3-phosphate, and the enzyme showed specificity for these four substrates only. It is proposed that the enzyme functions in cellular osmoregulation by providing glycerol 3-phosphate for the biosynthesis of glycerol, the main compatible solute in D. hansenii, and that the enzyme is well adapted to function in yeast cells exposed to osmotic stress.
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PMID:Purification and characterization of glycerol-3-phosphate dehydrogenase (NAD+) in the salt-tolerant yeast Debaryomyces hansenii. 197 35

The selenite-induced glutathione peroxidase in Chlamydomonas reinhardtii has been purified about 323-fold with a 10% yield, as judged by PAGE. The native enzyme had an Mr of 67,000 and was composed of four identical subunits of Mr 17,000. Glutathione was the only electron donor, giving a specific activity of 193.6 mumol/min per mg of protein. L-Ascorbate, NADH, NADPH, pyrogallol, guaiacol and o-dianisidine did not donate electrons to the enzyme. In addition to H2O2, organic hydroperoxides were reduced by the enzyme. The Km values for glutathione and H2O2 were 3.7 mM and 0.24 mM respectively. The enzyme reaction proceeded by a Ping Pong Bi Bi mechanism. Cyanide and azide had no effect on the activity. The enzyme contained approx. 3.5 atoms of selenium per mol of protein. On immunoprecipitation, Chlamydomonas glutathione peroxidase was precipitated and its activity was inhibited about 90% by the antibody raised against bovine erythrocyte glutathione peroxidase. The antibody also cross-reacted with the subunits of Chlamydomonas glutathione peroxidase in Western blotting SDS/PAGE. In terms of enzymic, physico-chemical and immunological properties, the experimental results demonstrate clearly that Chlamydomonas glutathione peroxidase resembles other well-characterized glutathione peroxidases from animal sources that contain selenium.
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PMID:Characterization and immunological properties of selenium-containing glutathione peroxidase induced by selenite in Chlamydomonas reinhardtii. 203 42

The presence of redox systems in microsomes of brown adipose tissue (BAT) in cold exposed rats was investigated and compared with liver. BAT microsomes showed high activity of lipid peroxidation measured both by the formation of malondialdehyde (MDA) and by oxygen uptake. NADH and NADPH dependent cytochrome c reductase activity were present in both BAT and liver microsomes. Aminopyrine demethylase and aniline hydroxylase activities, the characteristic detoxification enzymes in liver microsomes could not be detected in BAT microsomes. BAT minces showed very poor incorporation of [1-14C]acetate and [2-14C]mevalonate in unsaponifiable lipid fraction compared to liver. Biosynthesis of cholesterol and ubiquinone, but not fatty acids, and the activity of 3-hydroxy-3-methyl glutaryl CoA reductase appear to be very low in BAT. Examination of difference spectra showed the presence of only cytochrome b5 in BAT microsomes. In addition to the inability to detect the enzyme activities dependent on cytochrome P-450, a protein with the characteristic spectrum, molecular size in SDS-PAGE and interaction with antibodies in double diffusion test, also could not be detected in BAT microsomes. The high activity of lipid peroxidation in microsomes, being associated with large oxygen uptake and oxidation of NADPH, will also contribute to the energy dissipation as heat in BAT, considered important in thermogenesis.
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PMID:Microsomal redox systems in brown adipose tissue: high lipid peroxidation, low cholesterol biosynthesis and no detectable cytochrome P-450. 210 21

It was found that when Escherichia coli is grown in the presence of 0.2-0.3 mM menadione (2-methyl-1,4-naphthoquinone), an FMN-dependent NADH-quinone reductase increases more than 20-fold in the cytoplasmic fraction. The menadione-induced quinone reductase was isolated from the cytoplasmic fraction of induced cells. The purified enzyme had an Mr of 24 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme required flavin as a cofactor and a half-maximum activity was obtained with 0.54 microM FMN or 16.5 microM FAD. The enzyme had a broad pH optimum at pH 7.0-8.0 and reacted with NADH, but not with NADPH. The reaction followed a ping-pong mechanism and the intrinsic Km values for NADH and menadione were estimated to be 132 microM and 2.0 microM, respectively. Dicoumarol was a simple competitive inhibitor with respect to NADH with a Ki value of 0.22 microM. The electron acceptor specificity of this enzyme was very similar to that of NAD(P)H: (quinone acceptor) oxidoreductase (EC 1.6.99.2, DT-diaphorase) from rat liver. Since menadione is reduced by the two-electron reduction pathway to menadiol, the induction of this enzyme is likely to be an adaptive response of E. coli to partially alleviate the toxicity of menadione.
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PMID:Characterization of FMN-dependent NADH-quinone reductase induced by menadione in Escherichia coli. 211 86

The NADPH-dependent superoxide-generating oxidase of pig neutrophils is activated by sodium dodecyl sulfate in a cell-free system. The activation requires both membrane and cytosolic components. The membrane component was effectively extracted with 0.75% octyl glucoside and the extract was fractionated by wheat-germ-agglutinin-agarose column chromatography. The chromatography resulted in loss of the O2--generating activity in the cell-free system. The activity, however, was restored by the reconstitution with the fraction which passed through the column (fraction A) and the one eluted with N-acetylglucosamine (fraction B) using an octyl glucose dilution procedure: both fractions were pre-mixed in the presence of 0.75% octyl glucoside and diluted by putting the mixture into the detergent-free assay mixture. The latter fraction was copurified with cytochrome b558, the content of which is 2.12 +/- 0.53 nmol/mg protein (mean +/- SD, n = 5). The potency of fraction B in the reconstitution of the O2--generating activity was lost by heat treatment and decreased by protease treatment, whereas that of fraction A was not affected. Fraction A in the reconstitution of the O2--generating activity was replaced by lipid extracted from fraction A, furthermore, by exogenous phospholipid, azolectin. The O2--generating activity reconstituted with azolectin and the partially purified component in fraction B was dependent on SDS, cytosol and the concentrations of azolectin and FAD. The activity was sensitive to p-chloromercuribenzoate but not to azide. The maximal activity was obtained at pH 7.0-7.5. The Km values for NADPH and NADH were 0.024 mM and 0.57 mM, respectively. These properties were consistent with those of the NADPH oxidase responsible for the respiratory burst. The activity in the reconstitution system was 20.5 +/- 3.5 mumol O2-.min-1.mg-1 membrane-derived protein (mean +/- SD, n = 5) which shows that the membrane component was purified about 100-fold. These findings indicate that cytochrome b558 is probably a membrane component of the O2--generating NADPH oxidase and its activation in the cell-free system requires the reconstitution with phospholipids.
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PMID:Reconstitution of the partially purified membrane component of the superoxide-generating NADPH oxidase of pig neutrophils with phospholipid. 215 45

The superoxide generating NADPH oxidase was studied in an SDS-activated cell-free system. This system requires the participation of both membranal and cytosolic components. Cytosol derived from elicited peritoneal guinea pig macrophages was fractionated by several nucleotide affinity chromatography procedures. Various such fractionations led to the separation of two distinct factors, both of which are necessary for the activation and/or activity of the superoxide-forming NADPH oxidase. One factor (sigma 2), bound to octyl, 2',5'-ADP-, 5'-ATP-, 5'-GTP-agarose and carboxymethyl-Sepharose but did not bind to hexyl, 5'-AMP-, 5'-ADP- and 5'-GDP-agarose. The other factor (sigma 1) did not bind to any of the above matrices. Subsequent elution of sigma 2 from 2',5'-ADP-agarose was effected by ATP, GTP and NADPH but not by NADH. Elution from GTP-agarose was by ATP and GTP but not by NADPH. Elution from ATP-agarose was by ATP, GTP and also, albeit weakly, by NADPH. The above results suggest that sigma 2 contains a site which recognizes the phosphate group at the ribose 2' position in adenosine, and a site that recognizes purine nucleotide triphosphates.
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PMID:Nucleotide binding properties of cytosolic components required for expression of activity of the superoxide generating NADPH oxidase. 215 58

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diastereomeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C5-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (Mr) of the enzyme was estimated as 140,000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of Mr 35,000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.
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PMID:Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae. 222 22

A nucleoside catalyzing the oxidoreduction of NADH and K3Fe(CN)6 was isolated from Torula yeast RNA and also obtained in 0.05% yield by a series of steps: SDS-phenol extraction, nuclease P1 digestion, alkaline phosphatase digestion, anion exchange chromatography, and HPLC on an ODS column. Its chemical structure was clearly determined at 5-hydroxycytidine, from the results of FAB-MS and 1H and 13C NMR spectroscopies. The mass spectra, chromatographic behavior, UV spectra, and NMR spectra of this nucleoside from natural and synthetic sources were identical. This is the first report of an RNA catalyst having catalytic activity except for the cleavage and ligation of phosphodiester bonds of RNA. That an RNA has oxidoreduction activity indicates new possibilities for RNAs as "living molecules". 5-Hydroxycytidine may be a vestige of RNAs that formerly possessed metabolizing ability.
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PMID:A novel minimum ribozyme with oxidoreduction activity. 227 69

The flavoprotein DT-diaphorase (EC 1.6.99.2) is believed to play an important role in the body's defense system. This enzyme has been purified 13,000-fold with a recovery of 58% from a cytosolic fraction of abdominal fat obtained from an obese patient undergoing elective surgery. Purification of the enzyme to electrophoretic homogeneity was achieved after two chromatographic steps: (1) affinity chromatography on azodicumarol Sepharose 6B; (2) anion exchange chromatography on DEAE Sephacel. The enzyme exhibits a monomer molecular mass of 32 kDa in SDS-PAGE and has 1 FAD prosthetic group per 32 kDa monomer. The FAD prosthetic group appears to be firmly attached to the apoproprotein. The enzyme reduces azodyes and quinones and demonstrates a broad substrate specificity. The enzyme has characteristics that are similar to DT-diaphorase purified from rodent liver, especially the rat liver enzyme. Estimated Km values for NADH, NADPH and menadione are 200, 140 and 3.3 microM, respectively. Vmax values for these substrates in the same order are 762, 667 and 294 mumol/mg.min. Dicumarol and warfarin exhibited competitive inhibition with pyridine nucleotides. The inhibition constants (Ki) for the drugs were estimated to be 10 nM and 2.2 microM, respectively. When compared to several other tissues, abdominal fat has one of the highest DT-diaphorase activities (Martin, L.F., Patrick, S.D. and Wallin, R. (1987) DT-diaphorase in morbidly obese patients. Cancer Lett., 36, 341-347), but the specific role of the enzyme in human fat is unknown.
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PMID:Human DT-diaphorase, a potential cancer protecting enzyme. Its purification from abdominal adipose tissue. 246 Feb 16

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) were purified to a homogeneity from rat testis. The molecular weights of aldose reductase and aldehyde reductase were estimated to be 38,000 and 41,000 by SDS-polyacrylamide gel electrophoresis, and the pI values of these enzymes were found to be 5.3 and 6.1 by chromatofocusing, respectively. Aldose reductase had activity for aldo-sugars such as xylose, glucose and galactose, whereas aldehyde reductase was virtually inactive for these aldo-sugars. The Km values of aldose reductase for aldo-sugars were relatively high. When a correction was made for the fraction of aldo-sugar present as the aldehyde form, which is the real substrate of the enzyme, the Km values were much lower. Aldose reductase utilized both NADPH and NADH as coenzyme, whereas aldehyde reductase utilized only NADPH. Aldose reductase was activated significantly by sulfate ion, while aldehyde reductase was little affected. Both enzymes were inhibited strongly by the known aldose reductase inhibitors. However, aldehyde reductase was in general less susceptible to these inhibitors when compared to aldose reductase. Both aldose reductase and aldehyde reductase treated with pyridoxal 5-phosphate have lost the susceptibility to aldose reductase inhibitor, suggesting that in these two enzymes aldose reductase inhibitor interacts with a lysine residue.
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PMID:Characterization of aldose reductase and aldehyde reductase from rat testis. 250 Jan 52


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