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 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The complete carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin was elucidated through chemical and enzymatic methods including gas chromatography-mass spectrometry (GC-MS) and lectin affinity chromatography. Pronase digestion of thiostatin yielded a major glycopeptide fraction with asparagine the most abundant amino acid present. Based on one mole of aspartic acid, the following molar ratios obtained for the four major amino acids: aspartic acid (1.0), threonine (0.53), glycine (0.48) and serine (0.30). Neutral sugar analysis yielded a 3:2 molar ratio for mannose to galactose based on an assigned value to mannose of 3. On this basis, the fraction also contained 3 residues of sialic acid and, on average, 0 to 1 residue of fucose. GC-MS of partially methylated alditol acetates from the glycopeptide fraction identified the presence of biantennary and triantennary structure. Analyses of the neutral sugar and amino-acid composition, together with methylation data, support a biantennary N-linked structure for this major glycopeptide fraction and a triantennary N-linked structure as a lesser component. Sequencing of the desialyated 14C-labelled glycopeptide fraction by sequential exoglycosidase digestion and lectin affinity chromatography uncovered the following saccharide order: terminal galactose, N-acetylglucosamine and pentasaccharide inner core. This sequence is consistent with the N-linked glycan structures demonstrated by methylation and compositional analyses.
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PMID:The carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin. 794 64

Commonly a key element enabling proteins to function is an amino acid residue or residues with functional side chains having shifted pKa values. This article reports the results on a set of protein-based polymers (model proteins) that exhibit hydrophobic folding and assembly transitions, and that have been designed for the purpose of achieving large hydrophobic-induced pKa shifts by selectively replacing Val residues by Phe residues. The high molecular weight polypentapeptides, actually poly(tricosapeptides) with six varied pentamers in fixed sequence, were designed and synthesized to have the same amino acid compositions but different proximities between a single aspartic acid residue and 5 Phe residues per 30 residues. With the 5 Phe residues distal from the Asp residue, the observed pKa shift was 2.9 when compared to the Val-containing reference. With the 5 Phe residues within 1 nm of the Asp residue, the pKa shift was 6.2. This represents a free energy of interaction of 8 kcal/mole. To our knowledge, this is the largest pKa shift documented for an Asp residue in a polypeptide- or protein-water system. Data are reviewed that do not support the usual electrostatic arguments for pKa shifts of charge-charge repulsion and/or unfavorable ion self-energies arising from displacement of water by hydrophobic moieties, but rather the data are interpreted to indicate the presence of an apolar-polar repulsive free energy of hydration, which results from a potentially highly cooperative competition between apolar and polar species for hydration.
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PMID:Nanometric design of extraordinary hydrophobic-induced pKa shifts for aspartic acid: relevance to protein mechanisms. 805 71

The pH titration of nine amino acids (glycine, proline, valine, serine, glutamine, tryptophan, arginine, histidine and aspartic acid) in presence of urea in the concentration range 1-8 mole dm-3 has been performed. The results support suppression of the first dissociation constant (K1) of the amino acids and acceptance of H+ ions by the amide forming uronium ion (UH+). The second dissociation constant (K2) of the amino acids is affected relatively weakly by urea. Quantitative evaluation of different species existing in solution and the degree of dissociation of the acids as well as the degree of binding of H+ ion to the amide have been made. It has been found that the polarity of the aqueous-urea medium does not straight forwardly correlate with the altered pK1 of the amino acids. Urea can also affect the pH-titration behaviour of gelatin with an increase of the intrinsic pK of the acidic groups of the protein by 0.45 unit.
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PMID:Interaction of amino acids and gelatin with urea. 814 76

Erythrocyte agglutination by lectins from Allium sativum was inhibited only by mannose of the sugars tested. However, asialofetuin was more effective inhibitor of agglutination as compared to mannose. This led to the use of an asialofetuin-silica affinity column to isolate agglutinins of 110 and 25 kDa (ASA110 and ASA25). While ASA25 is a dimeric protein comprising of subunits of 12.5 and 13.0 kDa, ASA110 is a glycoprotein of two identical subunits of 47 kDa. ASA110 revealed to have a high content of aspartic acid, glycine, leucine and serine but low content of cysteine and methionine. It contains 14 residues of neutral sugars in addition to 43 residues of hexosamines per mole of lectin and requires metal ions for its functional conformation. Serological cross-reactions with other species showed some common epitopes of ASA110 and ASA25 present in A. porrum, A. ascalonicum, Narcissus alba, PHA and Con A but not in A. cepa. ASA110 with CHO cells indicated it to be weakly cytotoxic with LD50 of 160 microg/ml.
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PMID:A new high molecular weight agglutinin from garlic (Allium sativum). 904 16

The role of the HELLGH (residues 450-455) motif in the sequence of rat dipeptidyl peptidase III (EC 3.4.14.4) was investigated by replacing Glu451 with an alanine or an aspartic acid residue and by replacing His450 and His455 with a tyrosine residue by site-directed mutagenesis. Mutated cDNAs were expressed three or four times in Escherichia coli, and the resulting proteins were purified to apparent homogeneity. None of the expressed mutated proteins exhibited DPP III activity. The mutants of Glu451 contained 1 mol of zinc per mole of protein, but mutants His450 and His455 did not contain significant amounts of zinc as determined by atomic absorption spectrometry. The Leu453-deleted enzyme (having the zinc aminopeptidase motif HExxH-18-E) had almost the same order of binding affinity (for Arg-Arg-2-naphthylamide) as the wild-type enzyme, but the specificity constant was about 10%. These results provide evidence that the suitable number of amino acids included between Glu451 and His455 is three residues for the enzyme activity and confirm that residues His450, His455, and Glu451 are involved in zinc coordination and catalytic activity.
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PMID:The HELLGH motif of rat liver dipeptidyl peptidase III is involved in zinc coordination and the catalytic activity of the enzyme. 1038 75

In prion-related encephalopathies, the cellular prion protein (PrP(C)) undergoes a change in conformation to become the scrapie prion protein (PrP(Sc)) which forms infectious deposits in the brain. Conceivably, the conformational transition of PrP(C) to PrP(Sc) might be linked with posttranslational alterations in the covalent structure of a fraction of the PrP molecules. We tested a synthetic peptide corresponding to residues 106-126 of human PrP for the occurrence of spontaneous chemical modifications. The only asparagine residue, Asn108, was deamidated to aspartic acid and isoaspartic acid with a half-life of about 12 days. The same posttranslational modifications were found in recombinant murine full-length protein. On aging, 0.8 mol of isoaspartyl residue per mole of protein was detected by the protein-l-isoaspartyl methyltransferase assay (t(1/2) approximately 30 days). Mass spectrometry and Edman degradation of Lys-C fragments identified Asn108 in the amino-terminal flexible part of the protein to be partially converted to aspartic acid and isoaspartic acid. A second modification was the partial isomerization of Asp226' which is only present in rodents.
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PMID:Spontaneous deamidation and isomerization of Asn108 in prion peptide 106-126 and in full-length prion protein. 1044 69

Preparations of hypertensin II, obtained from the treatment of hypertensin I by the action of the hypertensin converting enzyme of plasma and purified by countercurrent distribution, were quantitatively analyzed for their amino acid content. Chromatography on ion exchange columns showed the presence of equimolar amounts of aspartic acid, proline, valine, isoleucine, tyrosine, phenylalanine, histidine, and arginine. Hypertensin I was found to contain one mole of leucine and one mole of histidine in addition to the amino acids of hypertensin II. These two amino acids were isolated from the conversion products of hypertensin I and identified as the peptide histidylleucine. Carboxypeptidase digestion of hypertensin I showed the carboxyl terminal sequence of amino acids to be residue-phenylalanyl-histidylleucine. Similar studies of hypertensin II demonstrated residue-phenylalanine. It was concluded that the conversion of hypertensin I by the plasma hypertensin converting enzyme involved hydrolysis of the phenylalanyl-histidine bond to form hypertensin II and histidylleucine. The further removal by carboxypeptidase of phenylalanine from hypertensin II destroyed all of the vasoconstrictor activity.
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PMID:The amino acid composition of hypertensin II and its biochemical relationship to hypertensin I. 1334 63

Monkey kidney cells tested in their first culture passage, 24 hours after their isolation from the animal host, required the same 13 amino acids for survival and growth as cell lines serially propagated in culture for years. Under the conditions of the present experiments, arginine, cystine, glutamine, histidine, and tyrosine proved necessary, over and above the 8 amino acids required for nitrogen balance in man. With the serially propagated lines, glutamic acid substituted for glutamine only at extremely high and non-physiological levels. In the monkey kidney cell cultures, however, glutamic acid and glutamine were interchangeable, mole for mole; and aspartic acid and asparagine were also effective as glutamine substitutes. Glycine was growth-stimulatory for monkey kidney cells in primary culture, and the cells grown in a glycine-deficient medium usually failed to survive subculture.
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PMID:The amino acid requirements of monkey kidney cells in first culture passage. 1352 76

1. The glycopeptides derived from a proteolytic digest of sialic acid-free alpha(1)-acid glycoprotein were separated on a DEAE-cellulose column into five main fractions. 2. The average molecular weight of these glycopeptides was 2400, except for one fraction whose molecular weight was 3100. The average molecular weight of the sialic acid-free carbohydrate units was found to be 2200. From these data and the carbohydrate content of the native protein and the assumed molecular weight of 44000, it was concluded that alpha(1)-acid glycoprotein probably possesses five carbohydrate units. The sialic acid-containing carbohydrate units of this glycoprotein have an average molecular weight of 3000, except for one unit the molecular weight of which is significantly higher. 3. The N-, non-N- and C-terminal amino acids of the main glycopeptides were determined. Aspartic acid and threonine occur in most peptides. Alanine, glycine, proline, serine and lysine were present in varying amounts. Traces of other amino acids were also found. 4. The amino acid sequence of three main glycopeptides was established and indicated that these glycopeptides are located at different positions of the polypeptide chain of the glycoprotein. These sequences are: Asp(NH(2))-Pro-Lys; Thr-Asp(NH(2))-Ala; Asp(NH(2))-Gly-Thr. 5. From the results of a series of chemical reactions (periodate oxidation, hydrazinolysis, dinitrophenylation, mild acid hydrolysis) it was shown that the hydroxyl group of the N-terminal threonine and the in-amino group of lysine are free and that the beta-carboxyl group of aspartic acid is present as amide. It was concluded that this amide group is involved in the carbohydrate-polypeptide linkages of at least four carbohydrate units of alpha(1)-acid glycoprotein. 6. The carbohydrate composition of the sialic acid-free glycopeptides was determined in terms of moles of neutral hexoses, glucosamine and fucose/mole. 7. Fucose, at least to the larger part, is not linked to sialic acid, and its (glycosidic) linkage is significantly more stable toward acid hydrolysis than the bond of the sialyl residues. 8. Heterogeneity of the carbohydrate units of alpha(1)-acid glycoprotein was found with regard to size and to content of fucose and sialic acid.
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PMID:THE CARBOHYDRATE-POLYPEPTIDE LINKAGES, THE AMINO ACID SEQUENCES OF THE PEPTIDES ADJACENT TO SOME OF THESE BONDS, AND THE COMPOSITION AND SIZE OF THE CARBOHYDRATE UNITS OF ALPHA-1-ACID GLYCOPROTEIN. 1434 11

Derivatives containing arginine-glycine-aspartic acid (RGD) inhibit fibrinogen binding to activated platelets and promote endothelial and smooth muscle cell attachment. An amphiphilic derivative of RGD that can be dissolved in an organic solvent has potential in the development of non-thrombogenic biomaterials. Such a derivative, LA-GRGD, was synthesised by coupling glycine-arginine-glycine-aspartic acid (GRGD) with lauric acid (LA). Its solubility and antithrombotic, cytotoxic and cell-binding effects were then evaluated in comparison with heparin (which is used clinically) and a fibronectin-engineered protein polymer (FEPP). Thromboelastography (TEG) was used to measure blood clotting time using fresh whole blood from healthy volunteers. Tissue factor (TF) activity was measured using plasma with a standard prothrombin time assay (PT). Cytotoxicity was assessed on human umbilical cord endothelial cells (HUVECs) using an Alamar blue assay. Solubility of the conjugate was assessed in a co-solvent. These techniques were used to study LA-GRGD, using heparin and FEPP as controls. The amphiphilic property of LA-GRGD was dependent on the feed mole ratio of GRGD to LA. LA-GRGD was soluble in acetone:water and water. LA-GRGD inhibited TF by >90% and prolonged TEG-r by 8.2+/-3.3 min (200 microg ml(-1)). Heparin inhibited TF by >90%, but prolonged TEG-r by 97.4+/-1.6 min (1 U ml(-1)); FEPP inhibited TF by >90% (100 microg ml(-1)) and prolonged TEG-r by 73.7+/-8.4 min (10 microg ml(-1)). Heparin had no cytotoxic effect on EC metabolism and viability at the concentrations studied (0.1-100 U ml(-1)). No significant cytotoxic effect was produced by LA-GRGD or FEPP at concentrations ranging from 0.1 microg ml(-1) to 50 microg ml(-1), but, at higher concentrations (100 microg ml(-1) and 200 microg ml(-1)), a detrimental effect was observed. Cell binding studies showed that LA-GRGD bound 29% of ECs compared with FEPP (60%) and heparin (22%). This new approach for synthesising amphiphilic RGD and its analogues has potential as a drug delivery system for the manufacture of new polymer formulations for use in bypass grafts and other tissue-engineered devices.
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PMID:Synthesis and evaluation of amphiphilic RGD derivatives: uses for solvent casting in polymers and tissue engineering applications. 1468 1


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