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)

Nonenzymatic glycation has been found to increase in a variety of proteins in diabetic patients. The present study examined a possibility of preventing glycation and subsequent structural modifications of proteins by alpha-lipoic acid (thioctic acid) as lipoate, a substance which has gained attention as a potential therapeutic agent for diabetes-induced complications. Incubation of bovine serum albumin (BSA) at 2 mg/ml with glucose (500 mM) in a sterile condition at 37 degrees C for seven days caused glycation and structural modifications of BSA observed by SDS-PAGE, near UV absorption, tryptophan and nontryptophan fluorescence, and fluorescence of an extrinsic probe, TNS (6-(p-toluidinyl)naphthalene-2-sulfonate). When BSA and glucose were incubated in the presence of lipoate (20 mM), glycation and structural modifications of BSA were significantly prevented. Glycation and inactivation of lysozyme were also prevented by lipoate. These results suggest a potential for the therapeutic use of lipoic acid against diabetes-induced complications.
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PMID:Lipoate prevents glucose-induced protein modifications. 145 92

Fast skeletal myosins were isolated from carp acclimated to 10 and 30 degrees C, and their structural and enzymatic properties were compared. Myosins in 0.5 M KCl were subjected to limited proteolysis by using various proteases including alpha-chymotrypsin, trypsin, and papain, and different SDS-PAGE patterns were seen for the 10- and 30 degrees C-acclimated myosins in all cases. Myosin subfragment-1 (S1) prepared from the 10 degrees C-acclimated myosin by alpha-chymotryptic digestion in 0.12 M NaCl showed higher acto-S1 Mg(2+)-ATPase activity and lower thermostability than S1 from the warm-acclimated myosin. The peptide maps and ATP-induced spectral changes of tryptophan fluorescence also showed an obvious difference between the two types of S1. Temperature acclimation further caused changes in the rod region of myosin, since the apparent sizes of light meromyosin were different from each other for the two types of myosin. Myosin from carp acclimated to 20 degrees C showed intermediate properties between those of the 10- and 30 degrees C-acclimated myosins. Myosin isoforms might be expressed in a temperature-dependent manner to compensate for the effect of seasonal environmental temperature variation on swimming ability.
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PMID:Fast skeletal myosin isoforms in thermally acclimated carp. 153 74

This work was undertaken in order to resolve some of the controversy in the literature concerning the properties of alpha-crystallins isolated in different laboratories. Bovine lens proteins were extracted and isolated by gel chromatography using 'Hoenders buffer' (0.02 M Tris-HCl, 1 mM EDTA, 80 mM NaCl, pH 7.3), 'Tardieu buffer' (0.04 M phosphate, 1 mM EDTA, 0.2 mM DTT, 0.06 M KCl, pH 6.8) and 'Thomson/Augusteyn' buffer (0.05 M Tris-HCl, 2 mM EDTA, 0.2 mM DTT, pH 8.0). The alpha-crystallin peaks were then divided into 12-16 pools and subjected to detailed physicochemical characterization. Fractionation by HPLC-GPC and quasi-elastic light scattering indicated that the size of the proteins decreased with increasing elution volume and that they were stable for at least 9 months at 20 degrees C. Molecular masses were found to range from over 2 mDa at the front of the peaks to around 600 kDa at the end. The size distributions, for the three buffers, were indistinguishable. No differences could be detected in the polypeptide distributions by SDS-PAGE. The proteins were also identical in their near- and far-UV circular dichroism spectra, accessibility of their sulphydryl groups to DTNB, tryptophan accessibility to quenching by acrylamide and iodide, and immunoreactivity with two monoclonal antibodies with different specificities. It is concluded that identical alpha-crystallins are isolated with the three different buffers and that variations in pH (6.9-8.0), ionic strength (60-150 mM) and cation (K, Na, Tris) during the isolation do not affect the properties of the protein. Claims that differing observations on the properties of alpha-crystallin may be attributed to the buffers used, are untenable.
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PMID:The effects of isolation buffers on the properties of alpha-crystallin. 155 51

In previous publications [e.g. Voskuilen, Vermond, Veeneman, Van Boom, Klasen, Zegers & Nieuwenhuizen (1987) J. Biol. Chem. 262, 5944-5946] we have shown that fibrin(ogen) chain fragment A alpha-(148-160) contains a site that contributes to the acceleration of Glu-plasminogen activation by tissue-type plasminogen activator (t-PA). In contrast with fibrin, this peptide, however, does not enhance the rate of mini-plasminogen activation. Therefore, possibly more stimulatory sites than A alpha-(148-160) are present in fibrin. In the present investigation we have localized a possible second type of stimulatory site in the fibrin(ogen) molecule. A whole CNBr digest of fibrinogen was applied to a Bio-Gel P-2 column run in water, pH 4. Two peaks with stimulatory activity were observed, one at the void volume and one between the void volume and the total volume. The former contained the previously described stimulating fragment FCB-2 [which comprises A alpha-(148-160)]; the latter had not been observed before and was characterized further. The stimulating material in the low-M(r) fraction of the Bio-Gel P-2 column was precipitated at pH 8.3 in a virtually pure form. It has a high tryptophan content, and an M(r) of 6500 as assessed by SDS/PAGE. On reduction, a main band of M(r) 2500 is seen, plus a weakly staining band of M(r) 4000. These properties plus the amino acid sequence data identify the fragment as FCB-5. FCB-5 consists of two chains, i.e. gamma-(311-336) and gamma-(337-379), linked by a single disulphide bond between Cys-gamma-326 and Cys-gamma-339. Both these chains and the disulphide bond appear to be essential for rate enhancement. FCB-5 enhances the activation rates of Glu-, mini- and micro-plasminogen, with all five kringles, only kringle V and without kringles respectively. FCB-5 binds t-PA, but none of the plasminogen forms binds to FCB-5. This indicates that the rate enhancements induced by FCB-5 are due to an effect on t-PA.
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PMID:Localization in the fibrinogen gamma-chain of a new site that is involved in the acceleration of the tissue-type plasminogen activator-catalysed activation of plasminogen. 156 67

Purified preparations of the Helminthosporium victoriae 190S (Hv190S) virus contain two sedimenting components that differ in capsid structure. The slower sedimenting component (190S-1) contained p88 and p83 as the major capsid proteins; the faster component (190S-2) contained p88 and p78. Previous peptide-mapping studies have shown the three capsid proteins to be closely related. Analysis by SDS-PAGE of in vivo-radiolabeled Hv190S virions indicated that 32P was predominantly incorporated in p88. Significantly less was detected in p83 and none in p78. Similar results were obtained in in vitro phosphorylation studies using [gamma-32P]ATP and purified 190S-1 and 190S-2. The in vitro results suggest that the Hv190S virions copurify with a protein kinase activity that catalyzes the transfer of gamma-phosphate from ATP to a target protein, presumably p78 in the 190S-2 virions and p83 in the 190S-1 component. Selective chemical cleavage at tryptophan residues of in vitro 32P-labeled capsid proteins revealed four labeled peptides among the cleavage products of both p83 and p88. Radioiodination studies with intact Hv190S virions indicated that p88 and p83, but not the nonphosphorylated p78, were accessible to iodination, suggesting that capsid protein phosphorylation entailed conformational changes.
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PMID:The Helminthosporium victoriae 190S mycovirus has two forms distinguishable by capsid protein composition and phosphorylation state. 158 40

Dimethylallyl tryptophan synthase (DMAT synthase) catalyzes the alkylation of L-tryptophan by dimethylallyl diphosphate to form 4-(gamma,gamma-dimethylallyl)-L-tryptophan. The enzyme from mycelia of Claviceps purpurea was purified approximately 125-fold to apparent homogeneity by chromatography on n-butyl Sepharose, Q Sepharose, phenyl Sepharose, and Protein Pak as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis by gel filtration chromatography and SDS-PAGE indicated that DMAT synthase is an alpha 2 dimer with a molecular mass of 105 kDa. The purified enzyme was active in metal-free buffer containing EDTA. However, activity was enhanced upon addition of divalent calcium or magnesium ions to the buffer. Values for KM and Vmax were determined in the metal-free EDTA buffer (KMDMAPP, 14 microM; KML-tryptophan, 40 microM; Vmax, 215 nmol min-1 mg-1), 4 mM CaCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 17 microM; Vmax, 504 nmol min-1 mg-1), and 4 mM MgCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 12 microM; Vmax, 455 nmol min-1 mg-1). The product was isolated and characterized by 1H NMR, uv, and FAB mass spectrometry.
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PMID:Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea. 160 39

1. The L-amino acid oxidase of the monocellate cobra (Naja naja kaouthia) venom was purified to electrophoretic homogeneity. The molecular weight of the enzyme was 112,200 as determined by Sephadex G-200 gel filtration chromatography, and 57,400 as determined by SDS-polyacrylamide gel electrophoresis. 2. The enzyme had an isoelectric point of 8.12 and a pH optimum of 8.5. It showed remarkable thermal stability, and, unlike many venom L-amino acid oxidase, was also stable in alkaline medium. The enzyme was partially inactivated by freezing. 3. The enzyme was very active against L-phenylalanine and L-tyrosine, moderately active against L-tryptophan, L-methionine, L-leucine, L-norleucine, L-arginine and L-norvaline. Other L-amino acids were oxidized slowly or not oxidized. 4. Kinetic studies suggest the presence of a side-chain binding site in the enzyme, and that the binding site comprises of at least four hydrophobic subsites.
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PMID:Purification and properties of the L-amino acid oxidase from monocellate cobra (Naja naja kaouthia) venom. 161 86

A prokaryotic expression plasmid, pKK-DT2, containing the cDNA of rat liver NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.99.2; DT-diaphorase) was constructed and used to transform Escherichia coli strain JM109. The rat liver quinone reductase was expressed in strain in JM109 and was inducible with isopropyl beta-D-thiogalactopyranoside (IPTG). The expressed rat protein was purified by affinity chromatography and had kinetic and physical properties identical with the protein purified from rat liver in that it could utilize either NADH or NADPH as the electron donor and its activity was inhibited by dicoumarol. In addition, we have generated four mutants, Arg-177----His (R177H), Arg-177----Ala (R177A), Arg-177----Cys (R177C) and Arg-177----Leu (R177L), using this expression system. Several of the mutants behaved anomalously on SDS/PAGE, but all of the mutant proteins had the expected M(r) as determined by electrospray m.s. These results and those obtained from enzyme kinetic analysis, u.v./visible absorption spectral analysis, and flavin and tryptophan fluorescence analysis of the wild-type enzyme and four mutants indicated that mutations at Arg-177 changed the conformation of the enzyme, resulting in a decrease in enzyme activity. Replacing Arg-177 with leucine altered the protein conformation and decreased FAD incorporation.
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PMID:Expression of rat liver NAD(P)H:quinone-acceptor oxidoreductase in Escherichia coli and mutagenesis in vitro at Arg-177. 162 1

Treatment of human beta 2 microglobulin (beta 2m) with defined oxygen-derived species generated by treatment with gamma-radiation was studied. As assessed by SDS/PAGE, the hydroxyl radicals (.OH) caused the disappearance of the protein band at 12 kDa that represents beta 2m, and cross-linked the protein into protein bands stable to both SDS and reducing conditions. However, when .OH was generated under oxygen in equimolar combination with the superoxide anion radical (O2.-), the high-molecular-mass protein products were less represented, and fragmented derivatives were not obviously detectable. Exposure to .OH alone, or to .OH + O2.- in the presence of O2, induced the formation of beta 2m protein derivatives with a more acidic net electrical charge than the parent molecule. In contrast, O2.- alone had virtually no effect on molecular mass or pI. Changes in u.v. fluorescence during .OH attack indicated changes in conformation, as confirmed by c.d. spectrometry. A high concentration of radicals caused the disappearance of the beta-pleated sheet structure and the formation of a random coil structure. Loss of tryptophan and significant production of dityrosine (2,2'-biphenol type) were noted, exhibiting a clear dose-dependence with .OH alone or with .OH + O2.-. The combination of .OH + O2.- induced a pattern of changes similar to that with .OH alone, but more extensive for c.d. and tryptophan oxidation (2 Trp/beta 2m molecule), and more limited for dityrosine formation. Lower levels of these oxidative agents caused the reproducible formation of species at 18 and 25 kDa which were recognized by antibodies against native beta 2m. These findings provide a model for the protein pattern observed in beta 2m amyloidosis described in the literature.
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PMID:Structural modifications of human beta 2 microglobulin treated with oxygen-derived radicals. 164 98

Alkaline phosphatase (ALP) is rapidly induced in the uterine subepithelial stroma after a natural or artificial decidual stimulus. During gestation ALP-specific activity peaked at Day 7 to 8 (Day 1 is day of detection of the copulation plug) followed by a rapid decline to control levels by Day 9. This elevation in enzyme activity was preceded by an 8-fold induction of a 2.6 kilobase (kb) mRNA. This mRNA was not preferentially localized to implantation sites. ALP activity was detected in the placenta at Day 9 and reached maximum specific activity at Day 19. The placental ALP was also encoded by a 2.6 kb mRNA. Uterine and placental ALPs were inhibited to the same extent by levamisole, L-tryptophan and homoarginine. The calculated Ki values for these inhibitors were not statistically different between the uterine and placental forms. Km values towards the substrate p-nitrophenylphosphate, however, were statistically different between the uterine and placental forms. Both uterine and placental ALPs were stimulated 3-4-fold by addition of 2 mM-Mg2+. Electrophoretic mobilities on SDS polyacrylamide gel, where the enzyme migrated as a single band, were the same. The uterine form, however, could be distinguished from the placental isoenzyme by separation on non-denaturing polyacrylamide gels; the uterine form had a single zone of activity which migrated with an intermediate mobility between the two zones of activity detected for the placental enzyme. These differences in mobility could be ascribed to the sialic acid content of the enzyme because treatment with neuraminidase resulted in the uterine and placental forms migrating with comparable but slower mobilities in non-denaturing gels.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Characterization and expression of uterine and placental alkaline phosphatases in the mouse. 169 26


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