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

2,4-Pentadienoyl-CoA reductase from Clostridium aminovalericum was purified to homogeneity (170-182 kDa). PAGE in the presence of SDS revealed a single band (44 kDa) indicating a homotetrameric structure. The native enzyme had a green colour and contained 0.4 mol FAD/subunit. Its unusual ultraviolet/visible-spectrum showed absorption maxima at 270, 402 and 715 nm as well as shoulders at 278, 360, 450 and 500 nm. Removal of the prosthetic group at pH 2 in the presence of salt and charcoal yielded a colourless and completely inactive apoenzyme, which could be reconstituted with FAD (not with FMN) to an active holoenzyme showing a normal flavoprotein spectrum (peaks at 369 nm and 436 nm). Thereby the FAD content increased to 0.9 mol/subunit with a concomitant rise in activity to 200% of the original value. Anaerobic reduction of the green enzyme by dithionite and reoxidation by air afforded a green preparation with a spectrum similar to that of the native enzyme. Addition of excess FAD to the green reductase also increased the activity by a factor of two. The green enzyme catalysed the oxidation of (E)-3-pentenoyl-CoA or (E)-3-hexenoyl-CoA to 2,4-pentadienoyl-CoA or 2,4-hexenoyl-CoA, respectively. 2-Pentenoyl-CoA or 4-pentenoyl-CoA were not oxidised. Meldola blue (8-dimethylamino-2,3-benzophenoxazine) and 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride (V = 26 nkat/mg protein) or ferricenium hexafluorophosphate (V = 1900 nkat/mg), but not NAD(P), served as electron acceptors. Reduction of 2,4-pentadienoyl-CoA (V = 370 nkat/mg) was observed with reduced benzyl viologen, but not with NAD(P)H as an electron donor. Although the enzyme had some pentenoyl-CoA delta-isomerase activity (1.2 nkat/mg), the only product of the reduction was 3-pentenoyl-CoA rather than 2-pentenoyl-CoA.
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PMID:A green 2,4-pentadienoyl-CoA reductase from Clostridium aminovalericum. 204 Feb 89

Lactate dehydrogenase (LDH) from white driving muscle of skate Raja clavata was purified by the differential precipitation of ammonium sulphate between 52 and 55% saturation. Only one protein band with the LDH activity was obtained by nondenaturing electrophoresis. The same result was obtained by the SDS-electrophoresis. The relative molecular weight calculated by this method in the presence of DS-Na was 34 kDa; Km was 29 +/- 7 and 71 +/- 16 microM for NAD.H and pyruvate, respectively. The reaction was maximally activated by 0.8-6.0 mM pyruvate and inhibited in the regions above this level. Dilution of LDH below concentration of 1 microgram/ml reduced the enzyme activity. The pH-optimum for the LDH activity ranged 7.0-8.0.
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PMID:[Isolation and characterization of lactate dehydrogenase from white fin muscles of the skate Raja clavata]. 208 91

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

Incubation of human erythrocyte membranes with [32P]NAD resulted in the label incorporation into the 37 kDa and 41 kDa proteins as determined by SDS-PAAG electrophoresis with subsequent autoradiography. Treatment of labeled membranes with HgCl2 caused a significant decrease of the protein band intensity in the autoradiograms. Incubation of purified GTP-binding protein from rat brain (Go-protein) with membranes and erythrocyte cytoplasm in the presence of [32P]NAD resulted in the label incorporation into the Go-protein alpha-subunit. This incorporation was markedly decreased after treatment of radiolabeled Go-protein with HgCl2. The data obtained testify to the existence in human erythrocytes of cytoplasmic and membrane-bound forms of cysteine-specific ADP-ribosyl transferase, for which the Go-protein serves as a substrate. The 37 kDa and 41 kDa proteins are substrates for the membrane-bound form of the enzyme.
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PMID:[Substrates of ADP-ribosyltransferase of human erythrocytes]. 212 Dec 86

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

Rat renal NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase was purified to apparent homogeneity in the present study. The purification included ammonium sulfate precipitation, DEAE-Sepharose CL-6B column chromatography, Blue Sepharose CL-6B column chromatography, Sephadex G-100 column chromatography and Mono-P isoelectrofocusing column chromatography. Among the chromatographies used, Mono-P chromatofocusing column of fast protein liquid chromatography gave the most powerful resolution. The enzyme was eluted at pH 6.75 on chromatofocusing column. It indicated that the rat renal enzyme is an acidic protein. Its molecular weight in SDS-polyacrylamide gel electrophoresis was 28 Kd, and the molecular weight of the enzyme having enzyme activity detected by gel filtration column chromatography was 55 Kd. For comparing the characteristics of rat renal enzyme with that of human placental enzyme, polyclonal antibodies and a monoclonal antibody against human placental enzyme were used. The rat renal enzyme did not react with the polyclonal antibodies of human placental enzyme, however, it reacted with a monoclonal antibody of human placental enzyme. The results indicated that the antigenicity of rat renal NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase is different from that of human placental enzyme. The amino acid composition of rat renal enzyme was also different from that of human placental enzyme. Nine tyrosine molecules were observed in the subunit of rat renal enzyme, while no tyrosine has been reported in human placental enzyme.
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PMID:Isolation of rat renal NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase. 225 Dec 93

Glucose-6-phosphate dehydrogenase (EC 1.1.1.49) has been purified from methanol grown Pseudomonas W6 by a simple procedure involving dye-ligand affinity chromatography on Cibacronblue F3G-A-Sephadex and Procion Red HE-3B-Sepharose. The purification procedure yielded a homogeneous enzyme with (1) high specific activity of 390 and 500 units/mg with NADP and NAD, respectively, and (2) low concentrations of contaminating activities. The molecular mass of the native enzyme was estimated to be 123 +/- 5 kDa. For the polypeptide chain after SDS denaturation a molecular mass of about 61 kDa was calculated. The kinetic behaviour of glucose-6-phosphate dehydrogenase exhibiting activity with either NADP or NAD was studied with respect to the substrate and coenzyme affinities and to ATP inhibition of enzyme activity. The applicability of prepared glucose-6-phosphate dehydrogenase as an auxiliary enzyme in clinical tests is discussed.
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PMID:Purification and characterization of glucose-6-phosphate dehydrogenase from Pseudomonas W6. 228 62

A NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) from porcine kidney was purified to homogeneity by acid precipitation, blue agarose affinity chromatography, hydroxyapatite-ultrogel adsorption chromatography, DEAE-Sephadex ion-exchange chromatography and NAD(+)-agarose affinity chromatography. The specific activity of the homogeneous enzyme was 31.2 U/mg. The molecular mass of the native enzyme was estimated to be 55,000 Da, whereas that of SDS-treated enzyme was 29,000 Da indicating that the native enzyme was dimeric. Compared to human placental 15-OH-PGDH, porcine kidney enzyme gave a similar general amino acid residue distribution. Chemical modification of the enzyme with N-ethyl maleimide (3 microM), N-chlorosuccinimide (20 microM) or 2,4,6-trinitrobenzenesulfonic acid (2.5 microM) followed pseudo-first-order inactivation kinetics, and inactivation could be prevented by the presence of NAD+ (1 mM) but not of prostaglandin E1 (140 microM) indicating the involvement of cysteine, methionine and lysine residues in the coenzyme binding site. Inactivation by diethyl pyrocarbonate (1.25 mM) or phenylglyoxal (10 mM) also showed pseudo-first-order kinetics suggesting that histidine and arginine residues were catalytically or structurally important in the native enzyme. These studies provide new insights into the structure and function of 15-OH-PGDH.
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PMID:Purification and characterization of NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase from porcine kidney. 239 68

Polyclonal, monospecific antibodies were produced against the two subunits (Mr 62,000, and Mr 31,000), isolated from the membrane-bound hydrogenase of Alcaligenes eutrophus H16. The antibodies (IgG fractions) were purified from crude sera by Protein A-Sepharose CL-4B chromatography. By double immunodiffusion assays and tandem-crossed immunoelectrophoresis the large and the small subunit were demonstrated not to be immunologically related. Immunological comparison of these subunits with the four non-identical subunits (Mr 63,000, 56,000, 30,000 and 26,000) of the NAD-linked, soluble hydrogenase from A. eutrophus H16 showed that the subunits of the membrane-bound hydrogenase did not cross-react with any of the antibodies raised against the four subunits of the NAD-linked enzyme and that, vice versa, none of these four subunits cross-reacted with antibodies raised against the two subunits of the membrane-bound hydrogenase. This means that A. eutrophus H16 contains altogether six non-identical immunologically unrelated hydrogenase polypeptides. The membrane-bound hydrogenases were isolated and purified from various aerobic H2-oxidizing bacteria: A. eutrophus H16, A. eutrophus type strain, A. eutrophus CH34, A. eutrophus Z1, A. hydrogenophilus, Paracoccus denitrificans and strain Cd2/01. All these proteins resembled each other and each consisted of two non-identical polypeptides. A complete separation of these subunits was achieved at high-yield by preparative FPLC gel filtration on three Superose 12 columns connected in series, using SDS and DTT-containing sodium phosphate buffer (pH 7.0). The small subunits of these enzymes turned out to be immunologically closely related to each other; they were either identical or almost identical. The large subunits were also related, but less pronounced. Only the large subunits from Z1 and type strain reacted fully identical with the H16 subunit. Of the two isolated, homogeneous subunits of the membrane-bound hydrogenase from A. eutrophus H16, the amino acid compositions and the NH2-terminal sequences have been determined. The results confirmed the diversity of the large and the small subunit. Furthermore, for comparison also the NH2-terminal sequences of the two subunits from the hydrogenase of A. eutrophus CH34 have been analysed.
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PMID:Immunological comparison of subunits isolated from various hydrogenases of aerobic hydrogen bacteria. 249 16


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