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)

Natural human interferon alpha 2 (IFN-alpha 2) was isolated from a preparation of partially purified human leucocyte IFN by monoclonal-antibody immunoaffinity chromatography. The purified protein had a specific activity of 1.5 x 10(8) i.u./mg; it was estimated to constitute 10-20% of the total antiviral activity of leucocyte IFN. N-Terminal amino-acid-sequence analysis identified the subspecies IFN-alpha 2b and/or IFN-alpha 2c, whereas IFN-alpha 2a was not detectable. The structure of natural IFN-alpha 2 was found to differ from that of its recombinant (Escherichia coli-derived) equivalent. First, reverse-phase h.p.l.c. showed that natural IFN-alpha 2 was significantly more hydrophilic then expected. Secondly, the apparent molecular mass of the natural protein determined by SDS/PAGE was higher than that of recombinant IFN-alpha 2; incubation under mild alkaline conditions known to eliminate O-linked carbohydrates resulted in a reduction of the apparent molecular mass to that of the recombinant protein. On sequence analysis of proteolytic peptides, Thr-106 was found to be modified. These results suggested that Thr-106 of natural IFN-alpha 2 carries O-linked carbohydrates. Reverse-phase h.p.l.c. as well as SDS/PAGE of natural IFN-alpha 2 showed that glycosylation is heterogeneous. For characterization of the carbohydrate moieties, the protein was treated with neuraminidase and/or O-glycanase and analysed by gel electrophoresis; in addition, glycopeptides obtained by proteinase digestion and separated by h.p.l.c. were characterized by sequence analysis and m.s. Further information on the composition of the glycans was obtained by monosaccharide analysis. The results indicate that natural IFN-alpha 2 contains the disaccharide galactosyl-N-acetylgalactosamine (Gal-GalNAc) linked to Thr-106. In part of the molecules, this core carbohydrate carries (alpha-)N-acetylneuraminic acid, whereas a disaccharide, probably N-acetyl-lactosamine, is bound to Gal-GalNAc in another proportion of the protein. Further glycosylation isomers are present in small amounts. As IFN-alpha 2 is the only IFN-alpha species with a threonine residue at position 106, it may represent the only O-glycosylated human IFN-alpha protein.
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PMID:Natural human interferon-alpha 2 is O-glycosylated. 204 76

L-Serine dehydratase with a specific activity of 15 nkat/mg protein was present in the anaerobic eubacterium Peptostreptococcus asaccharolyticus grown either on L-glutamate or L-serine. The enzyme was highly specific for L-serine with the lowest Km = 0.8 mM ever reported for an L-serine dehydratase. L-Threonine (Km = 22 mM) was the only other substrate. V/Km for L-serine was 500 times higher than that for L-threonine. L-Cysteine was the best inhibitor (Ki = 0.3 mM, competitive towards L-serine). The enzyme was purified 400-fold to homogeneity under anaerobic conditions (specific activity 6 mukat/mg). PAGE in the presence of SDS revealed two subunits with similar intensities (alpha, 30 kDa; beta, 25 kDa). The molecular mass of the native enzyme was estimated as 200 +/- 20 kDa (gel filtration) and 180 kDa (gradient PAGE). In the absence of oxygen the enzyme was moderately stable even in the presence of sodium borohydride or phenylhydrazine (5 mM each). However, by exposure to air the activity was lost, especially when the latter agent was added. The enzyme was reactivated by ferrous ion under anaerobic conditions. The inability of several nucleophilic agents to inactivate the enzyme indicated the absence of pyridoxal phosphate. This was confirmed by a microbiological determination of pyridoxal phosphate. However, the enzyme contained 3.8 +/- 0.2 mol Fe and 5.6 +/- 0.3 mol inorganic sulfur/mol heterodimer (55 kDa) indicating the presence of an [Fe-S] center. The enzyme was successfully applied to measure L-serine concentrations in bacterial media and in human sera.
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PMID:Purification and properties of an iron-sulfur-containing and pyridoxal-phosphate-independent L-serine dehydratase from Peptostreptococcus asaccharolyticus. 206 81

The present investigation evaluated amino acid utilization by 120 strains of Fusobacterium nucleatum in a chemically defined medium and attempted to relate the patterns to 3 proposed subspecies of F. nucleatum. Strains were inoculated into a chemically defined medium, with and without 2 g/l glucose, consisting of 14 inorganic salts, 21 amino acids, 23 vitamins and cofactors, and 7 purines and pyrimidines. After 7 days of anaerobic incubation, the spent culture medium, as well as the uninoculated control medium, were analyzed for amino acid content by ion chromatography. Amino acid utilization was determined by the differences in concentrations of amino acids found in inoculated and uninoculated samples. If greater than 34% of the amino acid was removed from the medium, the amino acid was considered to be utilized. Of the 21 amino acids present in the chemically defined medium, 8 amino acids, lysine, glutamine, asparagine, histidine, threonine, serine, glutamate and cysteine were consistently utilized. Four amino acids, tyrosine, tryptophan, methionine and aspartate were utilized by some strains but not others. Nine amino acids, alanine, leucine, isoleucine, glycine, valine, phenylalanine, proline, ornithine, and arginine were not utilized by any of the strains. The utilization patterns did not relate to subspecies formed on the basis of SDS-PAGE and DNA hybridization.
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PMID:Amino acid utilization by Fusobacterium nucleatum grown in a chemically defined medium. 208 74

The presence of potential N-linked glycosylation sites (Asn-X-Ser/Thr) in two forms of UDP glucuronosyltransferase, designated UDPGTr-2 and UDPGTr-4, has been deduced from cDNA sequence data. These forms were glycosylated when synthesized from expression vectors transfected into COS cells and were converted to faster migrating species on SDS polyacrylamide gels when treated with endoglycosidase H. The role of glycosylation was investigated by determining the substrate specificities and stabilities of the glycosylated enzymes and their unglycosylated variants which were synthesized in the presence of tunicamycin. Analysis of the activities towards 13 different aglycones showed that the glycosyl moiety was not essential for catalytic activity and had no effect on the substrate preference of each form. The stabilities of the proteins were not adversely affected by the absence of this posttranslational modification. A possible effect of N-linked oligosaccharides on the catalytic properties of these two forms of UDP glucuronosyltransferase is discussed.
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PMID:The effect of N-linked glycosylation on the substrate preferences of UDP glucuronosyltransferases. 210 15

On the basis of the datum that the level of Zn-alpha 2-glycoprotein (Zn alpha 2gp) in human seminal plasma was about 6-times higher than that in adult serum, Zn alpha 2gp was purified from fresh human seminal plasma approx. 70-fold with 60% yield over seminal plasma by DEAE-Sephacel, Zn-chelate Sepharose 4B and DEAE-5PW column chromatographies. The molecular weight of seminal plasma Zn alpha 2gp was 50,000 on Superose column chromatography, and 40,500 and 41,500 on SDS-polyacrylamide gel electrophoresis in the absence and presence of beta-mercaptoethanol, respectively. Plasma Zn alpha 2gp is a glycoprotein, while the protein from seminal plasma does not contain carbohydrate. The amino acid sequence of the first 17 residues of seminal plasma Zn alpha 2gp was Glu-Asn-Gln-Asp-Gly-Asn-Tyr-Ser-Leu-Thr-Tyr-Ile-Tyr-Thr-Gly-Leu-Ser. This sequence was completely identical with the amino acid residues from Glu-2 to Ser-18 in the N-terminal amino acid sequence of plasma Zn alpha 2gp. These data suggest that both Zn alpha 2gps in plasma and seminal plasma may be expressed from one gene, but their posttranslational modifications are different.
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PMID:Human seminal plasma Zn-alpha 2-glycoprotein: its purification and properties as compared with human plasma Zn-alpha 2-glycoprotein. 211 52

A soluble alkaline phosphatase was purified 10 000-fold in an overall yield of 8% from both of the cilia and cell bodies of the protozoan Paramecium tetraurelia. The concentration in cilia (1.7 microM) was 6-fold higher than in cell bodies, although the latter contained most of the activity due to their much greater volume. The purified protein showed a single (36 kDa) protein staining band on SDS-PAGE. This value, in conjunction with the apparent molecular mass of 66 kDa for the native enzyme (gel filtration) suggests a dimeric structure. The specific activity of the purified phosphatase ranged from 10 to 70 mumols.min-1.mg-1 at the pH-optimum of 8.0 and the Km for p-nitrophenyl phosphate was 81 microM. Basal enzyme activity was inhibited by metal chelators and stimulated up to 12-fold by addition of divalent cations. Mg2+ acted as a non-essential mixed-type activator with a half-maximal effect at 7 microM. Ca2+ was inhibitory, the extent of inhibition was dependent on the concentration of Mg2+ in the assay. Furthermore, the kinetics of inhibition by Ca2+ varied with the Mg2+ concentration. Phosphate, pyrophosphate, and SH-group blocking agents also strongly inhibited. The enzyme did not dephosphorylate Tyr- or Ser-/Thr-phosphoproteins. The Paramecium enzyme is not of lysosomal origin and its properties are quite different from all known phosphatases. It is a novel type of phosphatase since it (i) shows F(-)-inhibition like Ser/Thr-phosphatases but (ii) is inhibited by vanadate and molybdate like Tyr-phosphatases, and (iii) inhibition by Ca2+ has not been reported for any other phosphatase.
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PMID:Alkaline phosphatase from Paramecium cilia and cell bodies: purification and characterization. 215 27

Bovine plasma protein C inhibitor was purified; it was then characterized in comparison with human protein C inhibitor. The specific inhibitory activity of the purified inhibitor for bovine activated protein C was 8,500 times that of the inhibitor in plasma. The purified inhibitor showed a single band with Mr 56,000 by SDS-PAGE at pH 7.0, and two bands at pH 8.8, a major one with Mr 56,000 and a minor one with Mr 105,000, under both unreduced and reduced conditions. The pI range of the inhibitor was between 4.4 and 6.1. The Mr of the inhibitor was reduced by treatment with neuraminidase, O-glycanase, and also with glycopeptidase-A, suggesting that the inhibitor has both Asn-linked and Ser/Thr-linked carbohydrate chains. Twenty-seven of the NH2-terminal 49 amino acid residues of the bovine inhibitor, which lacks the first 4 residues from the NH2-terminal amino acid sequence of human inhibitor, were identical to those of the human inhibitor. The bovine inhibitor inhibited bovine and human activated protein C, human thrombin, Factor Xa, Factor XIa, and plasma kallikrein with Ki = 1.0, 5.2, 2.6, 3.0, 1.3 X 10(-8) M, and 4.5 X 10(-9) M, respectively. The inhibitory rates for activated protein C and thrombin were accelerated significantly in the presence of heparin or negatively charged dextran sulfate. However, the acceleration by heparin or dextran sulfate for the inhibition of Factor Xa, Factor XIa, and plasma kallikrein was not significant. The bovine inhibitor did not inhibit human Factor XIIa or plasmin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Bovine plasma protein C inhibitor with structural and functional homologous properties to human plasma protein C inhibitor. 216 Apr 49

THR1, the gene from Saccharomyces cerevisiae, encoding homoserine kinase, one of the threonine biosynthetic enzymes, has been cloned by complementation. The nucleotide sequence of a 3.1-kb region carrying this gene reveals an open reading frame of 356 codons, corresponding to about 40 kDa for the encoded protein. The presence of three canonical GCN4 regulatory sequences in the upstream flanking region suggests that the expression of THR1 is under the general amino acid control. In parallel, the enzyme was purified by four consecutive column chromatographies, monitoring homoserine kinase activity. In SDS gel electrophoresis, homoserine kinase migrates like a 40-kDa protein; the native enzyme appears to be a homodimer. The sequence of the first 15 NH2-terminal amino acids, as determined by automated Edman degradation, is in accordance with the amino acid sequence deduced from the nucleotide sequence. Computer-assisted comparison of the yeast enzyme with the corresponding activities from bacterial sources showed that several segments among these proteins are highly conserved. Furthermore, the observed homology patterns suggest that the ancestral sequences might have been composed from separate (functional) domains. A block of very similar amino acids is found in the homoserine kinases towards the carboxy terminus that is also present in many other proteins involved in threonine (or serine) metabolism; this motif, therefore, may represent the binding site for the hydroxyamino acids. Limited similarity was detected between a motif conserved among the homoserine kinases and consensus sequences found in other mono- or dinucleotide-binding proteins.
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PMID:Yeast homoserine kinase. Characteristics of the corresponding gene, THR1, and the purified enzyme, and evolutionary relationships with other enzymes of threonine metabolism. 216 4

Treatment of PC12 cells with either nerve growth factor (NGF), a differentiating factor, or epidermal growth factor (EGF), a mitogen, resulted in 7-15-fold activation of a protein kinase activity in cell extracts that phosphorylated microtubule-associated protein (MAP) 2 on serine and threonine residues in vitro. Both the NGF-activated kinase and the EGF-activated kinase could be partially purified by sequential chromatography on DEAE-cellulose, phenyl-Sepharose and hydroxylapatite, and were identical with each other in their chromatographic behavior, apparent molecular mass (approximately 40 kDa) on gel filtration, substrate specificity, and phosphopeptide-mapping pattern of MAP2 phosphorylated by each kinase. Moreover, both kinases were found to be indistinguishable from a mitogen-activated MAP kinase previously described in growth-factor-stimulated or phorbol-ester-stimulated fibroblastic cells, based on the same criteria. Kinase assays in gels after SDS/polyacrylamide gel electrophoresis revealed further that the NGF- or EGF-activated MAP kinase in PC12 cells, as well as the EGF-activated MAP kinase in fibroblastic 3Y1 cells resided in two closely spaced polypeptides with an apparent molecular mass of approximately 40 kDa. In addition, these MAP kinases were inactivated by either acid phosphatase treatment or protein phosphatase 2A treatment. These results indicate that MAP kinase may be activated through phosphorylation by a differentiating factor as well as by a mitogen. MAP kinase activation by EGF was protein kinase C independent; it reached an almost maximal level 1 min after EGF treatment and subsided rapidly within 30-60 min. On the other hand, NGF-induced activation of MAP kinase was partly protein kinase C dependent and continued for at least 2-3 h.
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PMID:Microtubule-associated-protein (MAP) kinase activated by nerve growth factor and epidermal growth factor in PC12 cells. Identity with the mitogen-activated MAP kinase of fibroblastic cells. 217 61

Bacteriophage T7 expresses a serine/threonine-specific, cAMP-independent protein kinase activity encoded by the early gene 0.7. The phosphoproteins specifically resulting from gp0.7 protein kinase expression in T7-infected Escherichia coli have been examined by one-dimensional, SDS-polyacrylamide gel electrophoresis. Only seven major, stable phosphoproteins dependent on gp0.7 protein kinase expression are observed. Two of the gp0.7 protein kinase-specific phosphoproteins observed have been previously identified: the beta' subunit of RNA polymerase and the RNA processing enzyme RNase III. The gp0.7-catalyzed protein phosphorylation activity appears at 9-11 min postinfection at 30 degrees. The new phosphoproteins have a metabolic stability comparable to that of uninfected cell phosphoproteins. T7 protein kinase expression causes the phosphorylation of the same, limited set of proteins in B, C, or K strains of E. coli. Expression of the T3 and BA14 phage protein kinase activities also produces the same phosphoproteins.
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PMID:Protein kinase of bacteriophage T7 induces the phosphorylation of only a small number of proteins in the infected cell. 218 69


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