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)

An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.
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PMID:Purification and characterization of an NAD(P)H:quinone oxidoreductase from Glycine max seedlings. 760 72

The properties of a mouse liver copper binding protein (CuBP) and human placental S-adenosylhomocysteine hydrolase (SAHH) were compared to test the hypothesis that CuBP is SAHH. CuBP and SAHH migrated identically on SDS-polyacrylamide gel electrophoresis gels, and their 48-kDa monomers both self-associate to tetramers. Human placental SAHH cross-reacted with polyclonal antibodies to mouse liver CuBP, and CuBP from mouse liver cross-reacted with two monoclonal antibodies to human placental SAHH. A third monoclonal antibody to human placenta SAHH reacted weakly with the mouse liver protein but well with CuBP from human lymphoblasts. NAD(+)-activated CuBP has high SAHH enzymatic activity. Moreover, human placental SAHH, like mouse liver CuBP, has a single high affinity copper binding site per 48-kDa subunit. Thus, the data confirm that CuBP is SAHH, and SAHH is proposed to be a bifunctional protein with roles in sulfur-amino acid metabolism and copper metabolism. The copper binding activity of SAHH is proposed to play a significant role in the intracellular distribution of copper, and SAHH enzymatic activity may influence copper metabolism through its role in cysteine biosynthesis from methionine.
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PMID:Identification of a major hepatic copper binding protein as S-adenosylhomocysteine hydrolase. 765 50

Deoxyhypusine synthase is the first enzyme involved in the post-translational formation of hypusine, a unique amino acid that occurs at one position in a single cellular protein, eukaryotic translation initiation factor 5A (eIF-5A). This NAD-dependent enzyme catalyzes the formation of deoxyhypusine by transfer of the butylamine portion of spermidine to the epsilon-amino group of a specific lysine residue in the eIF-5A precursor. Its purification from rat testis was accomplished by ammonium sulfate fractionation and successive ion-exchange chromatographic steps, followed by chromatofocusing on a hydrophilic resin (Mono P). A pI of 4.7 was determined by isoelectric focusing. Amino acid sequences of five tryptic peptides of the pure enzyme did not correspond to any sequences in the protein data banks. The enzyme migrates as a single band on SDS-polyacrylamide gel electrophoresis with an apparent monomer molecular mass of approximately 42,000 Da. Matrix-assisted laser desorption mass spectrometry gave a monomer mass of 40,800 Da. There is evidence, however, that the active enzyme exists as a tetramer of this subunit. Rabbit polyclonal antibodies to the 42-kDa protein precipitated deoxyhypusine synthase activity. The enzyme shows a strict specificity for NAD. Purified deoxyhypusine synthase catalyzes the overall synthesis of deoxyhypusine and, in the absence of the eIF-5A precursor, catalyzes the cleavage of spermidine.
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PMID:Deoxyhypusine synthase from rat testis: purification and characterization. 772 68

Integrin alpha 7 is a major substrate in skeletal muscle cells for the cell surface, glycosylphosphatidylinositol-anchored, arginine-specific ADP-ribosyltransferase. Since ADP-ribosylarginine hydrolase, the enzyme responsible for cleavage of the ADP-ribosylarginine bond and a component with the transferase of a putative ADP-ribosylation cycle, is cytosolic, the processing of ADP-ribosylated integrin alpha 7 was investigated. Following incubation of differentiated mouse C2C12 myoblasts with [adenylate-32P]NAD and analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, two [32P]ADP-ribosylated forms of integrin alpha 7 were resolved. By pulse-chase and purification of the radiolabeled proteins on a laminin affinity column, it was demonstrated that a 105-kDa ADP-ribosylated form originated from a mono-ADP-ribosylated 102-kDa form and represented integrin alpha 7 modified at more than one site. The additional site(s) of modification, utilized at higher NAD concentrations, were located in the 63-kDa N-terminal segment of integrin alpha 7. Both [32P]ADP-ribosylated integrins were loosely associated with the cytoskeleton, bound to laminin affinity columns, and immunoprecipitated with antibodies to integrin beta 1. 32P label was rapidly removed from [32P]ADP-ribosylated integrin alpha 7 at either site of modification, a process inhibited by free ADP-ribose or p-nitrophenylthymidine-5'-monophosphate, an alternative substrate of 5'-nucleotide phosphodiesterase. The processed integrin alpha 7 was unavailable for subsequent ADP-ribosylation, although the amount of surface integrin alpha 7 remained constant. During the processing, no loss of label was observed from integrin alpha 7 radiolabeled with [14C]NAD, containing 14C in the nicotinamide proximal ribose, consistent with degradation of the ADP-ribose moiety by a cell surface 5'-nucleotide phosphodiesterase. Thus, cell surface ADP-ribosylation, in contrast to intracellular ADP-ribosylation, is not readily reversed by ADP-ribosylarginine hydrolase and seems to operate outside the postulated ADP-ribosylation cycle.
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PMID:Processing of ADP-ribosylated integrin alpha 7 in skeletal muscle myotubes. 772 41

Efficient fluorescence energy transfer from aromatic residues to the pyridine moiety of the bound coenzyme (NAD) of UDP-glucose 4-epimerase from Kluyveromyces fragilis had been reported earlier (Mukherji, S., and Bhaduri, A. (1992) J. Biol. Chem. 267, 11709-11713). We have employed N-bromosuccinimide (NBS) to identify tryptophan as the exclusive aromatic donor in the energy transfer. The characteristic UV absorption spectrum associated with Trp oxidation is observed during NBS modification of two of the four Trp residues of native epimerase along with concomitant inactivation of the enzyme. Excellent correlation between the observed inactivation and abolition of fluorescence energy transfer to coenzyme from Trp in epimerase upon treatment with NBS implicates the involvement of the same two tryptophans in both catalytic activity and fluorescence energy transfer. SDS-polyacrylamide gel electrophoresis and fluorescence data preclude gross structural/conformational changes in epimerase due to NBS oxidation. The susceptible tryptophans do not reside at the substrate binding site as substrates and UMP fail to protect against NBS modification. However, failure of sodium borohydride to reduce the bound NAD in the NBS-inactivated epimerase suggests that the reactive tryptophans are close to the coenzyme. Tryptophan fluorescence lifetime values of 1.9 and 3.9 ns for the native and 3.5 ns for the NBS-modified epimerase, complemented by a linear Stern-Volmer plot (effective Stern-Volmer constant = 2.85 M-1) of acrylamide quenching, suggest that the two key tryptophans are buried close to an intrinsic quencher, presumably NAD.
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PMID:Two tryptophans at the active site of UDP-glucose 4-epimerase from Kluyveromyces fragilis. 774 74

During growth on n-tetradecane a novel NADH-dependent carbonyl reductase is induced in the Gram-positive bacterium Rhodococcus erythropolis (Peters, P., Zelinski, T. and Kula, M.R. (1992) Appl. Microbiol. Biotechnol. 38, 334-340). The enzyme has been purified to homogeneity using fractional pH precipitation, anion exchange chromatography and affinity chromatography. The isoelectric point of the oxidoreductase is 4.4. The apparent molecular mass of the native enzyme is 161 kDa, that of the subunits 40 kDa as determined by SDS gel electrophoresis. A tetrameric structure of the carbonyl reductase is consistent with these results. Important biochemical data concerning the application of the reductase are: a broad pH-optimum, temperature optimum at 40 degrees C and stability at room temperature for more than 5 days. The oxidoreductase accepted as substrate aliphatic and aromatic ketones, keto esters (esters of keto carboxylic acids) and halogenated carbonyl compounds and reduced them to the corresponding hydroxyl compounds with (S)-configuration with more than 98% enantiomeric excess. The NAD(+)-dependent oxidation of primary alcohols was not catalyzed by the carbonyl reductase, whereas secondary alcohols and hydroxy acid esters were oxidized to the corresponding carbonyl compounds at about 10-fold slower reaction rates compared to the reduction.
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PMID:Purification and characterization of a novel carbonyl reductase isolated from Rhodococcus erythropolis. 776 39

A small protein with high affinity for homologous DNA was isolated from Sulfolobus solfataricus homogenate by mineral acid extraction. It was purified using a two-step procedure including CM-cellulose and RP-HPL chromatographies. The protein was electrophoretically homogeneous, had a molecular weight of 7.147 kDa and an amino acid composition with a high content of lysine and glutamic acid residues. The protein was able to protect DNA against thermal denaturation and DNAse I digestion in a dose-dependent manner. After incubation of the sulfolobal homogenate in the presence of 32P-NAD, followed by the purification steps, the protein was modified by ADPribose, as revealed by reaction product analysis, SDS-PAGE and autoradiography.
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PMID:In the thermophilic archaeon Sulfolobus solfataricus a DNA-binding protein is in vitro (Adpribosyl)ated. 788 65

The key enzyme of tyrosine biosynthesis in many Gram-negative prokaryotes is cyclohexadienyl dehydrogenase. The Zymomonas mobilis gene (tyrC) coding for this enzyme was cloned in Escherichia coli by complementation of a tyrosine auxotroph. The tyrC gene was 882 bp long, encoding a protein with a calculated molecular mass of 32086 Da. The Z. mobilis cyclohexadienyl dehydrogenase expressed in E. coli was purified to electrophoretic homogeneity. The subunit molecular mass of the purified enzyme was 32 kDa as determined by SDS/PAGE. The ratio of the activity of arogenate dehydrogenase to that of prephenate dehydrogenase (approximately 3:1) remained constant throughout purification, and the two activities were therefore inseparable. The genetic and biochemical data obtained demonstrated a single enzyme protein capable of catalyzing either of two reactions. Km values of 0.25 mM and 0.18 mM were obtained from prephenate and L-arogenate, respectively. The Km value obtained for NAD+ (0.09 mM) was the same regardless of whether the enzyme was assayed as arogenate dehydrogenase or as prephenate dehydrogenase. Unlike the corresponding enzyme of Pseudomonas aeruginosa or E. coli, the cyclohexadienyl dehydrogenase of Z. mobilis lacks sensitivity to feedback inhibition by L-tyrosine. A typical NAD(+)-binding domain was found to be located at the N-terminus of the protein. Although the deduced amino-acid sequence of the Z. mobilis cyclohexadienyl dehydrogenase showed relatively low identity (19-32%) with the prephenate dehydrogenases of Bacillus subtilis and Saccharomyces cerevisiae, as well as with the cyclohexadienyl dehydrogenase components of the bifunctional T-proteins of E. coli and Erwinia herbicola, a presumptive motif was identified which may correspond to critical residues of the binding site for cyclohexadienyl substrate molecules. Immediately upstream of tryC a portion of a gene was sequenced and found to exhibit clearcut homology of the deduced amino-acid sequence with the B. subtilis hisH gene product. Thus, the Zymomonas gene organization is reminiscent of the linkage of genes encoding a tryosine-pathway dehydrogenase and a histidine-pathway aminotransferase in B. subtilis.
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PMID:An allosterically insensitive class of cyclohexadienyl dehydrogenase from Zymomonas mobilis. 791 85

The gene coding for the glycosomal glyceraldehyde-3-phosphate dehydrogenase from Leishmania mexicana has been cloned into vector pET3A and expressed as a soluble and active protein in Escherichia coli BL21(DE3) in which the endogenous gene has been inactivated by mutation. The recombinant enzyme was purified to near homogeneity by ammonium sulphate precipitation, followed by hydrophobic and cation-exchange chromatography. From a 1-L culture of E. coli cells, 25 mg purified protein was obtained with a specific activity of 125 units/mg. The recombinant protein restores the natural E. coli phenotype when expressed at low level. The enzyme has also been partially purified from glycosomes of cultured L. mexicana promastigotes. The recombinant and the native proteins show identical mobilities on SDS/PAGE, and have the same isoelectric point and similar pH-activity profiles. The kinetics of both enzymes are very similar, the most important aspect being their lower apparent affinity for the cofactor NAD when compared to all other homologous enzymes studied, with the exception of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei.
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PMID:Purification and characterization of the native and the recombinant Leishmania mexicana glycosomal glyceraldehyde-3-phosphate dehydrogenase. 792 31

Nicotinamide and 3-aminobenzamide prevent TNF-alpha-mediated cytotoxicity, indicating that ADP-ribosylation plays a crucial role in this reaction. We have studied the role of ADP-ribosylation during TNF-alpha action in TNF-alpha-sensitive and TNF-alpha-resistant cells. Treatment of 3T3 cells with TNF-alpha, in the presence of [adenylate-32P]NAD followed by SDS-PAGE, revealed the involvement of specific ADP-ribosylation of a 90-kDa protein in TNF-alpha-mediated cytotoxicity. The stability of the ADP-ribosyl linkage on the 90 kDa protein in 100 mM 2-(N-cyclohexylamino)ethanesulfonic acid at pH 9.0 confirmed that ADP-ribosylation of the 90 kDa protein was mediated by an enzymatic reaction. Analysis of ADP-ribose residues by phosphodiesterase hydrolysis showed that the 90-kDa protein was modified by poly ADP-ribosylation. Poly ADP-ribosylation of the 90-kDa protein concomitant with cytotoxicity was observed in all TNF-alpha-sensitive but not TNF-alpha-resistant cells. Inhibition of ADP-ribosylation of the 90-kDa protein by benzamide but not by benzoic acid abrogated cytotoxicity, which further suggested that the poly-ADP-ribosylation of the 90-kDa protein is causally related to TNF-alpha-induced cell death. Our results demonstrate that TNF-alpha modifies a specific protein by poly-ADP-ribosylation during its action. Furthermore, ADP-ribosylation of specific proteins may be yet another mechanism regulating protein function during cellular metabolism.
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PMID:Poly ADP-ribosylation of a 90-kDa protein is involved in TNF-alpha-mediated cytotoxicity. 802 88


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