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)

Human erythrocyte aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) was purified to apparent homogeneity. The native enzyme has a molecular weight of about 210,000 as determined by gel filtration, and SDS-polyacrylamide gel electrophoresis of this enzyme yields a single protein and with a molecular weight of 51,500, suggesting that the native enzyme may be a tetramer. The enzyme has a relatively low Km for NAD (15 microM) and a high sensitivity to disulfiram. Disulfiram inhibits the enzyme activity rapidly and this inhibition is apparently of a non-competitive nature. In kinetic characteristic and sensitivity to disulfiram, erythrocyte aldehyde dehydrogenase closely resembles the cytosolic aldehyde dehydrogenase found in the liver of various species of mammalians.
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PMID:Purification and partial characterization of aldehyde dehydrogenase from human erythrocytes. 22 30

A new method of isolating the microsomal ghost fractions has been developed. The membranes were purified from adsorbed protein, ribosomes and intravesicular content. The purified membranes contained 6 times less RNA than did the original microsomes. The phospholipid:protein ratio in the ghosts increased from 0.34 to 0.80. Protein electrophoretic pattern ghost was more homogenous than that of microsomes. SDS-polyacrylamide electrophoresis showed that the ghosts contain 11 fractions as compared to 21 in microsomes. A high degree of membrane purification did not affect the inactivation of microsomal enzymic systems. Practically all cytochrome P-450 in the ghosts was found in its active form. The activities of NAD(P)H-dependent systems of dimethylaniline and ethylmorphine demethylation, p-hydroxylation of aniline were well retained. The removal of non-membrane protein allows to obtain an electrone microscope picture of the lipid bilayer as a structural basis of microsomal membrane.
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PMID:[Isolation, analysis and characterization of microsomal ghost fractions]. 85 99

The Escherichia coli haemoglobin-like flavohaemoprotein (Hmp) has been purified to near homogeneity using two chromatographic steps. The prosthetic groups are identified as FAD and protohaem IX. SDS/PAGE has indicated a molecular mass of 44 kDa for the monomeric protein consistent with the amino-acid sequence deduced from the hmp+ gene. The protein, as isolated, is in the Fe(III) state, exhibiting absorbance maxima at 403.5, 540 (shoulder) and 627 nm. The ferrous and carbonmonoxyferrous states resemble those of haemoglobin, showing maxima at 431.5 and 558 nm, and 421, 542 and 566 nm respectively. Upon aerobic addition of NAD(P)H, the ferric state is reduced to the oxygenated Fe(II) state, characterized by maxima at 413, 544 and 580 nm. This oxy form is not stable and slowly decays to the ferric state. Addition of dithionite and nitrite to the ferric protein results in the formation of a nitrosyl complex, whose e.p.r. characteristics indicate that the b-type haem is attached to the protein through a nitrogenous ligand, probably originating from a histidine residue.
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PMID:Spectroscopic studies on an oxygen-binding haemoglobin-like flavohaemoprotein from Escherichia coli. 133 13

Site-directed mutagenesis was utilized to identify binding sites for NAD(P)H and dicumarol in rat liver NAD(P)H:quinone oxidoreductase (NQOR, EC 1.6.99.2). The mutant cDNA clones were generated by a procedure based on the polymerase chain reaction and were expressed in Escherichia coli. The mutant enzymes were purified to apparent homogeneity as judged by SDS-polyacrylamide gel electrophoresis and were found to contain 2 FADs/enzyme molecule identical with that of the wild-type NQOR. Purified mutant enzymes Y128D, G150F, G150V, S151F, and Y155D showed dramatic decreases in activities in the reduction of dichlorophenolindophenol in comparison with the activities of the wild-type enzyme, whereas the activities of F124L, T127V, T127E, Y128V, Y128F, S151A, and Y155V were similar to those of NQOR. Enzyme kinetic analysis revealed that the Km values of T127E, Y128D, G150F, G150V, S151F, and Y155D were, respectively, 4-, 2-, 13-, 5-, 26-, and 19-fold higher than the Km of NQOR for NADPH, and were, respectively, 2-, 3-, 7-, 3-, 20-, and 11-fold higher than that of NQOR for NADH. The kcat values of Y128D, G150F, and G150V were also much lower than those of NQOR, but the kcat values of other mutants were similar to those of the wild-type enzyme. The Km values of the mutants for dichlorophenolindophenol were the same or slightly higher than that of NQOR. The apparent inhibition constants (Ki) for dicumarol on Y128V and F124L were elevated 12 and 8 times, respectively. Similar, but smaller, changes on Ki for 4-hydroxycoumarin were also observed. This study demonstrated that residues Gly150, Ser151, and Tyr155 in the glycine-rich region of NQOR are essential for NADPH and NADH binding and Tyr128 is important for dicumarol binding. Based on the results of the study, it is proposed that the glycine-rich region of the enzyme, along with other residues around the region, forms a beta sheet-turn-alpha helix structure important for the binding of the pyrophosphate group of NADPH and NADH.
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PMID:Identification of a glycine-rich sequence as an NAD(P)H-binding site and tyrosine 128 as a dicumarol-binding site in rat liver NAD(P)H:quinone oxidoreductase by site-directed mutagenesis. 138 97

A plasmid has been constructed by cloning the complete crtI gene encoding phytoene desaturase from Erwinia uredovora behind the lac Z promoter of pUC18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus. This plasmid mediated the overexpression of phytoene desaturase in transformed Escherichia coli. The overexpressed enzyme was sequestrated into inclusion bodies requiring urea treatment for solubilization. Purification to homogeneity was subsequently performed on a DEAE-cellulose column and by SDS-polyacrylamide gel electrophoresis. The purification scheme allowed the isolation of 5.3 mg of homogeneous desaturase protein from 100 ml of E. coli cell suspension. On SDS-polyacrylamide gel electrophoresis an apparent molecular mass of 56.2 kDa was determined. An antiserum raised against phytoene desaturase cross-reacted with the expressed protein and was employed to monitor the isolation steps. Upon removal of urea, desaturase activity was restored. The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene. FAD was involved in desaturation, whereas NAD and NADP were inhibitory. This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state.
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PMID:Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase. 140 Mar 5

Protein kinase N (PKN) is a protein kinase rapidly activated by nerve growth factor (NGF) and other agents in PC12 pheochromocytoma and additional cell types. PKN is selectively inhibited by purine analogs, and this property has served both as a diagnostic for PKN activity and to establish its apparent involvement in certain pathways of the NGF mechanism of action. The present work has focused on further characterization, identification, and purification of NGF-activated PKN. We show here that PKN can be substantially enriched by elution from ion exchange resins with ATP. We exploited this novel technique (nucleotide affinity exchange chromatography) to devise two alternative isolation schemes for PKN. One utilizes sequential chromatographic steps and provides a preparation that is apparently 60% homogeneous for PKN and represents a total enrichment of approximately 10,000-fold. The other is a single column procedure and includes prewashes with NAD. This method yields material that is about 5-10% homogeneous for PKN, requires about 1 h, and can be applied to multiple samples in parallel. The ATP elution technique furthermore distinguishes NGF-regulated from basal PKN activity and thereby suggests the presence of distinct PKN isoforms. The applications of sucrose gradient centrifugation, gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/silver staining, affinity labeling with 8-azido-ATP/SDS-PAGE, and autophosphorylation (after SDS-PAGE, blotting and renaturation) all indicate that PKN has an apparent molecular mass of 45-47 kDa and is mainly monomeric in solution. These and additional properties appear to distinguish PKN from many previously described protein kinases.
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PMID:Nerve growth factor-activated protein kinase N. Characterization and rapid near homogeneity purification by nucleotide affinity-exchange chromatography. 140 Apr 78

The NAD-dependent glycerol-3-phosphate dehydrogenase (glycerol-3-phosphate:NAD+ oxidoreductase; EC 1.1.1.8; G3P DHG) was purified 178-fold to homogeneity from Saccharomyces cerevisiae strain H44-3D by affinity- and ion-exchange chromatography. SDS-PAGE indicated that the enzyme had a molecular mass of approximately 42,000 (+/- 1,000) whereas a molecular mass of 68,000 was observed using gel filtration, implying that the enzyme may exist as a dimer. The pH optimum for the reduction of dihydroxyacetone phosphate (DHAP) was 7.6 and the enzyme had a pI of 7.4. NADPH will not substitute for NADH as coenzyme in the reduction of DHAP. The oxidation of glycerol-3-phosphate (G3P) occurs at 3% of the rate of DHAP reduction at pH 7.0. Apparent Km values obtained were 0.023 and 0.54 mM for NADH and DHAP, respectively. NAD, fructose-1,6-bisphosphate (FBP), ATP and ADP inhibited G3P DHG activity. Ki values obtained for NAD with NADH as variable substrate and FBP with DHAP as variable substrate were 0.93 and 4.8 mM, respectively.
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PMID:Purification and characterization of glycerol-3-phosphate dehydrogenase of Saccharomyces cerevisiae. 149 20

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH) is a key enzyme involved in the catabolism of the prostaglandins. The cDNA for human placental 15-PGDH has been expressed in Escherichia coli as a catalytically active protein. The polymerase chain reaction was used to introduce restriction endonuclease sites at each end of the 15-PGDH coding sequence. The 15-PGDH DNA was then inserted into the bacterial expression plasmids pUC-18 and pUC-19 which contain the isopropyl-l-thio-beta-D-galactopyranoside (IPTG) inducible lacZ promoter. Extracts from E. coli containing these expression plasmids exhibited 15-PGDH activity which was inducible with (IPTG). Crude extracts from E. coli expressing 15-PGDH activity were found to contain proteins of the predicted sizes in stained SDS-polyacrylamide gels and in Western blots using human placental 15-PGDH antiserum. The specific activity in E. coli extracts was several hundred-fold higher than that seen in extracts from human placenta.
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PMID:Expression of the cDNA for NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase as a catalytically active enzyme in Escherichia coli. 150 55

1. In diapausing eggs of the silkworm, Bombyx mori, activity of NAD-sorbitol dehydrogenase (EC 1.1.1.14, SDH) is almost negligible, but is increased by acclimation at 5 degrees C (Yaginuma et al., 1990, J. comp. Physiol. B160, 277-285). To elucidate the mechanism regulating SDH activity, the following experiments were conducted. Anti-SDH serum was made in a mouse using purified sheep liver SDH. 2. This antiserum reacted with Bombyx egg SDH purified partially by Blue Sepharose CL-6B and Sephacryl S-300 column chromatographies. 3. SDS-PAGE and immunoblotting analyses using the antiserum showed that SDH activity was correlated with the amount of the enzyme protein. 4. These results indicate that biosynthesis of SDH is induced by acclimation at 5 degrees C in diapause eggs of B. mori.
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PMID:Biosynthesis of NAD-sorbitol dehydrogenase is induced by acclimation at 5 degrees C in diapause eggs of the silkworm, Bombyx mori. 152 25

Mutants resistant to 3-aminobenzamide, a known inhibitor of ADP-ribosyltransferase, were obtained from Streptomyces griseus IFO 13189, a streptomycin-producing strain. One (strain no. 4), which had significantly reduced ADP-ribosyltransferase activity, was analysed in detail. Mutant 4 displayed a conditional phenotype with respect to cultivation temperature. At 30 degrees C, it exhibited severely reduced ability to produce aerial mycelium (on solid medium) and submerged spores and streptomycin (in liquid culture), but this ability was fully restored at 25 degrees C. The mutant produced A-factor normally, regardless of cultivation temperature, and exhibited normal ability to accumulate ppGpp intracellularly. SDS-PAGE analyses of cellular proteins labelled by [32P]NAD revealed that an ADP-ribosylated protein with a molecular size of 44 kDa, which appeared in sporulating cultures of the parent strain, was missing from the mutant grown at the non-permissive temperature (30 degrees C). Genetic analysis showed that the aba mutation conferring resistance to 3-aminobenzamide was tightly linked to the altered phenotype. Failure to ADP-ribosylate certain cellular protein(s), presumably due to the aba mutation, may be responsible for impaired differentiation in this mutant.
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PMID:The possible role of ADP-ribosylation in sporulation and streptomycin production by Streptomyces griseus. 152 13


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