Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

An immunopeptide bearing a3 allotypic determinant(s) was isolated from the gamma chain of an a3 homozygous rabbit (G222-2) immunized with type III pneumococcal vaccine. Immunocogical properties of peptides were studied using a radioimmunoassay that involved inhibition by these peptides of a reaction between 125I-labeled anti-a3 antibody and Sepharose-bound a3 immunoglobulin G (IgG). The gamma chain was isolated from IgG of restricted heterogeneity and then citraconylated and digested with trypsin. The tryptic digest (TD1) was passed through an anti-a3 immunoabsorbent column either directly or after an intermediate step of Sephadex G-75 chromatography. The bound peptides (T1) were eluted with 0.1 M acetic acid and further digested with trypsin. The digest (TD2) was again run on the anti-a3 immunoabsorbent column to purify the bound immunopeptide T2. In the radioimmunossay this immunopeptide was found to have major a3 determinant(s). Its molecular weight was found to be approximately 6,000, which decreased to about 3,000 after reduction and alkylation. These data, together with NH2- and COOH-terminal analyses and cysteine peptide mapping, demonstrated that T2 is composed of two polypeptide chains linked by a disulfide bond, one from the cysteine 22 region having lysine at the COOH terminus and the other from the cysteine 92 region arginine at the COOH terminus. The lysine peptide was separated from the arginine peptide and its NH2-terminal sequence was found to be Gly-Asx-Glx-Ser-Thr-Cys. Since the cysteine is at position 22, the lysine peptide starts at position 17. It has approximately 22 residues. The framework sequence from 17 to 20 is different from those reported so far. In addition, the heavy chain used in these studies has some other unusual features including a histidine, probably in the first hypervariable region. The presence of histidine in the first hypervariable region of rabbit heavy chain has not been reported previously. The other peptide which is about 30 amino acids in length and ends with arginine 94, probably includes positions 67, 70, 71, 84, and 85 that are believed to have substitutions correlating with a allotypes. In a hypothetical three-deminsional model of the Fv portion of rabbit anti-SIII antibody BS-5, residues 17 to 33 of the lysine peptide and 67 to 79 and 84 to 85 which may be present in the arginine peptide are fully exposed on the surface and are far removed from the antibody combining site.
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PMID:Studies on the structural localization of rabbit H chain allotypic determinants controlled by the a locus. Purification and immunological properties of an immunopeptide bearing a3 allotypic determinants. 6 65

Digestion with trypsin of monoclonal rat IgG of IgG1 and IgG2a subclasses produced two fragments, isolated only in dissociating media. The larger fragment (mol. wt. 120,000 Da) was comprised of the two light chains covalently bound to shortened gamma chains. Amino acid sequence of the shortened gamma chain indicated that the site of cleavage is located at the beginning of the C gamma 1 domain at a position homologous to residue 139 of mouse gamma heavy chain of IgG1 MOPC 21. The smaller fragment (mol. wt. 13,000 Da) was found to consist of the entire variable domain of the heavy chain and probably a short stretch of the C gamma 1 domain. The unique susceptibility of rat monoclonal IgG1 and IgG2a is likely to be the result of the presence of a lysine residue in a loop of C gamma 1 domain, which therefore is accessible to trypsin. Tryptic cleavage of rat monoclonal antibodies of IgG1 and IgG2a subclasses can be considered as a simple method to produce a fragment related to the VH domain.
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PMID:Isolation and characterization of a VH domain fragment from monoclonal rat IgG of IgG1 and IgG2a subclasses. 249 34

The fetal hemoglobin of 137 Dutch newborn has been examined by peptide mapping after digestion with trypsin, in order to detect the T gamma chain (gamma chain bearing a replacement Ile replaced by Thr at position 75) and to assess its quantity relative to the total gamma chains. Forty-two (31%) individuals were carriers of this variant, with an average amount of T gamma chain equal to 18.89% +/- 4.12 (1 SD). Three babies had T gamma levels higher than 30%, outside, therefore, the upper limit of the distribution. If we consider these three children homozygous carriers and assume that the remaining thirty-nine are heterozygotes, the gene frequency of T gamma in this population is 0.16 and the observed frequencies of the three phenotypes (T gamma-/-, T gamma+/- and T gamma +/+) agree closely with the expected ones. The occurrence and levels of T gamma chain are apparently not subject to variations during the last 25 weeks of gestation. In a group of twenty-nine fetuses investigated between the 12th and 20th week gestation, seven were, in fact, positive and showed T gamma percentages (12-26%) within the limits found in newborn. Also, the relative amounts of G gamma and A gamma chains were identical to those calculated in babies born at term. The composition of fetal hemoglobin remains, thus, uniform from the 12th week of gestation to birth. A comparison of the distribution of T gamma and A gamma percentages in T gamma positive newborn supports indirectly the identification of T gamma chain as a polymorphic variant of A gamma chain.
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PMID:Frequency and quantitative levels of T gamma chain (Hb F Sardinia) in the fetal hemoglobin of newborn and fetuses. 615 2

Binding of fibrinogen to human platelets depends on the interaction of the gamma-chain carboxy-terminal segment with specific receptors exposed by different agonists such as ADP, epinephrine, and thrombin. The functions of a series of synthetic peptides encompassing the sequence of the 15 carboxy-terminal residues of the gamma chain were investigated in this study. Both pentadecapeptide (gamma 397-411) and dodecapeptide (gamma 400-411) inhibited binding of 125I-fibrinogen to ADP-treated platelets, with the concentration causing 50% inhibition (IC50) being 28 microM. In comparison, decapeptide (gamma 402-411) was almost 4 times less active (IC50 = 106 microM), thus suggesting that the two histidine residues (gamma 400-401) are required for a full inhibitory effect. A heptapeptide (gamma 405-411) had a similar effect (IC50 = 102 microM) whereas a pentapeptide (gamma 407-411) was even less inhibitory (IC50 = 190 microM), indicating that the lack of lysine (gamma 406) further diminishes the reactivity of the platelet recognition site on the gamma chain of human fibrinogen. The heptapeptide (gamma 400-406) containing two histidine residues and derived from the dodecapeptide by proteolytic degradation with trypsin had very low inhibitory activity. The synthetic peptides inhibited fibrinogen-supported platelet aggregation in the same order of decreasing reactivity: pentadecapeptide = dodecapeptide greater than decapeptide = heptapeptide greater than pentapeptide. Modified synthetic pentadecapeptides bearing tyrosine or cysteinyltyrosine at the amino terminal were prepared to provide a means for radiolabeling and for formation of molecules of higher valency.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Platelet receptor recognition site on human fibrinogen. Synthesis and structure-function relationship of peptides corresponding to the carboxy-terminal segment of the gamma chain. 632 8

Two types of normal human plasma fibrinogen--peak 1 and peak 2--are distinquishable by DEAE-cellulose gradient elution chromatography. The elution characteristics of peak 2 fibrinogen, which amounts to about 15% of the total, are attributable to the presence of a gamma chain variant, gamma', which is more negatively charged than gamma chains and makes up about half of all such chains in that peak [Mosesson M. W., Finlayson, J. S. & Umfleet, R. A. (1972), J. Biol. Chem. 247, 5223-5227]. Analyses of reduced S-carboxymethylated fibrin that had first been incubated in the presence of Factor XIIIa plus the fluorescent amine donor dansylcadaverine (DNScad) showed that the same amount of this compound could be incorporated covalently into either type of gamma chain. Furthermore, the DNScad-labeled COOH-terminal CNBr fragment (CNBr e) derived from the S-carboxymethylated gamma chain was smaller than the DNScad-labeled fragment (CNBr e') from the gamma' chain (Mr, 3200 and 4900) by about the same amount as the difference in size between the respective parent chains (Mr, 49,400 and 51,500). DNScad-CNBr e or DNScad-cNBR e' could be further cleaved by trypsin to yield a smaller fluorescent fragment corresponding to the penultimate tryptic gamma chain peptide containing the DNScad-glutamine acceptor and lysine donor crosslinking functions. The COOH-terminal amino acids of gamma and gamma' chains were valine and leucine, respectively. The rates of Factor XIIIa-catalyzed crosslinking of peak 1 and peak 2 fibrin were the same, but peak 1 fibrin gamma chains formed only one species of crosslinked dimer (gamma gamma) whereas peak 2 fibrin gamma chains yielded three (gamma gamma, gamma gamma', gamma'gamma'). We conclude that gamma' chains are functionally normal but have an extended COOH-terminal sequence accounting for their more negative charge and larger size relative to gamma chains.
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PMID:Human plasma fibrinogen heterogeneity: evidence for an extended carboxyl-terminal sequence in a normal gamma chain variant (gamma'). 693 47

Biochemical studies of fibrin cross-linking were conducted to identify the specific Aalpha chain lysine residues that potentially serve as Factor XIIIa amine donor substrates during alpha polymer formation. A previously characterized Factor XIIIa fibrin lysine labeling system was employed to localize sites of donor activity based on their covalent incorporation of a synthetic peptide acceptor substrate analog modelled after the NH2-terminal cross-linking domain of alpha2 antiplasmin. Peptide-decorated fibrin was prepared using purified fibrinogen as the starting material. Cyanogen bromide digestion, immunoaffinity chromatography, high pressure liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (anti-peptide) methodologies were employed to isolate purified CNBr fibrin fragments whose structures included the acceptor probe in cross-linked form and, therefore, represented regions of (amine) donor activity. Five alpha chain CNBr fragments (within Aalpha 208-610) and one gamma chain CNBr fragment (gamma 385-411) were the only portions of fibrin found associated with the acceptor peptide, based on collective sequencing, mass, and compositional data. Trypsin digestion, HPLC, and enzyme-linked immunosorbent assay (anti-peptide) methodologies were used to isolate smaller derivatives whose structures included an alpha chain tryptic cleavage product (the donor arm) cross-linked to the trypsin-resistant synthetic peptide (the acceptor arm). Biochemical characterization and quantitative peptide recovery data revealed that 12 of the 23 potential lysine donor residues within alpha 208-610 had incorporated the peptide probe, whereas gamma chain donor activity was due solely to peptide cross-linking at (gamma) Lys406; the alpha chain lysines, Lys556 and Lys580, accounted for 50% of the total alpha chain donor cross-linking activity observed, with Lys539, Lys508, Lys418, and Lys448 contributing an additional 28% and Lys601, Lys606, Lys427, Lys429, Lys208, Lys224, and/or Lys219 responsible for the remaining proportion (2-5%, each). The collective findings extend current models proposed for the mechanism of alpha polymer formation, raise questions concerning the physiological role of multiple alpha chain donor sites, and, most importantly, provide specific information that should facilitate future efforts to identify the respective lysine and glutamine partners involved in native fibrin alpha chain cross-linking.
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PMID:Identification of the alpha chain lysine donor sites involved in factor XIIIa fibrin cross-linking. 870 12

Elevated plasma fibrinogen levels are a major risk factor for thrombosis. This report shows two mechanisms by which fibrinogen can affect the fibrinolysis rate in vitro and thus may lead to thrombosis. First, the lysis rate of fibrin decreases as the initial concentration of fibrinogen increases. Second, a minor variant form of fibrinogen decreases the rate of fibrinolysis. This variant, gammaA/gamma' fibrinogen, has one altered gamma chain and is known to bind to factor XIII zymogen. In a fibrinolysis assay containing purified thrombin, fibrinogen, tissue-type plasminogen activator, and plasminogen, clots from gammaA/gammaA and gammaA/gamma' fibrinogen lysed at similar rates. However, when factor XIII was added, slower lysis was seen in gammaA/gamma' fibrin clots when compared with gammaA/gammaA fibrin clots. A D-dimer agglutination assay showed that the gammaA/gamma' clots were more highly cross-linked than the gammaA/gammaA clots. The lysis rates of gammaA/gamma' clots were similar to gammaA/gammaA clots in the presence of N-ethylmaleimide, a specific inhibitor of factor XIIIa. The gammaA/gamma' fibrin clots made in the presence of factor XIII showed increased proteolytic resistance to both plasmin and trypsin. Clots made from afibrinogenemic plasma reconstituted with gammaA/gamma' fibrinogen also showed significant resistance to lysis compared with gammaA/gammaA fibrinogen. These data demonstrate gammaA/gamma' fibrin is resistant to fibrinolysis, possibly as a result of concentrating factor XIII on the clot. The total fibrinogen concentration and the amount of gammaA/gamma' fibrinogen increase clot stability in vitro and thus may contribute independently to the risk of thrombosis in humans.
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PMID:Resistance of gammaA/gamma' fibrin clots to fibrinolysis. 916 58

Hydrophobic membrane proteins often have complex functions and are thus of great interest. However, their analysis presents a challenge because they are not readily soluble in polar solvents and often undergo aggregation. We present a sequential CNBr and trypsin in-gel digestion method combined with mass spectrometry for membrane protein analysis. CNBr selectively cleaves methionine residues. But due to the low number of methionines in proteins, CNBr cleavage produces a small number of large peptide fragments with MWs typically >2000, which are difficult to extract from gel pieces. To produce a larger number of smaller peptides than that obtained by using CNBr alone, we demonstrate that trypsin can be used to further digest the sample in gel. The use of n-octyl glucoside (n-OG) to enhance the digestion efficiency and peptide recovery was also studied. We demonstrate that the sensitivity of this membrane protein identification method is in the tens of picomole regime, which is compatible to the Coomassie staining gel-spot visualization method, and is more sensitive than other techniques reported in the literature. This CNBr/trypsin in-gel digestion method is also found to be very reproducible and has been successfully applied for the analysis of complex protein mixtures extracted from biological samples. The results are presented from a study of the analysis of bacteriorhodopsin, nitrate reductase 1 gamma chain, and a complex protein mixture extracted from the endoplasmic recticulum membrane of mouse liver.
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PMID:Development and applications of in-gel CNBr/tryptic digestion combined with mass spectrometry for the analysis of membrane proteins. 1458 51

In order to better understand the mechanisms governing display of mast cell characteristics in human myeloid cells, we have studied the mast cell phenotype in human promyelocytic (HL-60) and myelocytic (U-937, TPH-1) vs. basophilic (KU-812) and mast cell (HMC-1) lines, in part also in skin mast cells and blood monocytes, at mRNA and protein level before and after stimulation with mast cell growth factors. In unstimulated cells, mRNA for the stem cell factor (SCF) receptor c-kit and the gamma chain of the high-affinity IgE receptor (FcepsilonRI) was noted in all cells studied. Like mast and basophilic cells, THP-1 cells expressed the FcepsilonRIalpha and beta chains and weakly histidine decarboxylase (HDC), but they lacked mRNA for mast cell-specific proteases [tryptase, chymase, carboxypeptidase A (CPA)]. In contrast, HL-60 and U-937 cells lacked FcepsilonRIalpha, but expressed tryptase and chymase, HL-60 cells also CPA. KU-812 cells failed to express the basophil-specific marker 2D7. After a 10-day culture with SCF or fibroblast supernatants, baseline mRNA expression of most mast cell characteristics was upregulated, whereas c-kit mRNA expression decreased in all but THP-1 cells. Differential mRNA expression of FcepsilonRI vs. protease (tryptase) was confirmed at protein level by immunocytochemistry and enzymatic activity. KU-812 cells are thus closest to skin mast cells in that they express all molecules studied, except for chymase, followed by THP-1 cells that lack all mast cell proteases. In contrast, HL-60 and U-937 cells fail to express the FcepsilonRIalpha and beta chains but express most mast cell proteases. The selective and differential expression of mast cell characteristics in human myeloid cell lines suggests that induction of the mast cell phenotype is regulated by several independent genes and that mast cells and basophils branch off at early and distinct points of myeloid development.
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PMID:Differential expression of mast cell characteristics in human myeloid cell lines. 1533 53