Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

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

The intracellular distribution of a membrane-bound protein disulfide isomerase (PDI) in rat liver was studied by quantitative immunoprecipitation, and its microsomal localization was confirmed. The content of the enzyme was 1 to 2% of total microsomal protein, and it was almost equally distributed between rough and smooth microsomes. The enzyme was not solubilized from microsomes by high concentrations of KCl, but was readily solubilized by detergents. Since PDI in microsomes was susceptible to digestion by trypsin, at least some parts of the enzyme molecule are exposed on the outside surface of microsomal vesicles. However, the binding of antibodies to microsomal PDI and the modification of the glutamine residues of PDI molecules by transglutaminase suggested that the molecules are not extensively exposed on the surface. Solubilized PDI was unable to rebind to microsomes or to become incorporated into reconstituted membrane of detergent-solubilized microsomes, showing that the association of this enzyme with the membrane is not simply mediated by hydrophobic interaction.
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PMID:Intracellular and intramembranous localization of a protein disulfide isomerase in rat liver. 728 43

The primary aim of these studies was to identify and biochemically characterize GTP-binding proteins in the nucleus. We found that an 80 kDa protein was responsible for the majority of the GTP-binding activity detected in rabbit liver nuclear preparations as assayed by photoaffinity labeling with [alpha-32P]GTP. The GTP-binding activity was partially extracted only after treatment of nuclear envelope preparations with 0.5 M NaCl and 1% Triton-X 100, which suggested that this GTP-binding protein was a component of the nuclear pore/lamina fraction. The Triton-X-100/NaCl-solubilized 80 kDa protein was purified by a series of steps that included DEAE-Sephacel, Mono-Q, and Ultrogel AcA34 chromatographies. Microsequence analysis of two peptides generated by trypsin digestion of the 80 kDa protein indicates that it shares sequence similarity with the tissue transglutaminases. Purified preparations of the 80 kDa protein show a Ca(2+)-stimulated transglutaminase activity, as assayed by the incorporation of [3H]putrescine into caesin, which is strongly inhibited by GTP but not by GDP. A 36 kDa GTP-binding protein copurified with the 80 kDa GTP-binding protein through all of the chromatography steps and sequence analysis suggests that the 36 kDa protein represents a proteolytic fragment of the amino-terminal half of the 80 kDa protein and thus serves to mark the GTP-binding domain within the 80 kDa protein. The 36 kDa fragment has a significantly higher efficiency of [alpha-32P]GTP incorporation compared to the 80 kDa protein, suggesting that the carboxyl-terminal half of the GTP-binding protein/transglutaminase imparts a negative constraint on GTP-binding activity or on the subsequent incorporation of radiolabeled GTP.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification and biochemical characterization of an 80 kilodalton GTP-binding/transglutaminase from rabbit liver nuclei. 749 18

In vivo, articular chondrocytes produce an important amount of extracellular matrix (cartilage) whose quality is impaired upon inflammation or aging leading to arthritis or arthrosis. Transglutaminases (EC 2.3.2.13) are a family of enzymes which have been shown to be involved in extracellular matrix stabilization, cell differentiation and possibly in initiation and propagation of inflammatory diseases. It is therefore of interest to study transglutaminase activity in chondrocytes. Transglutaminase activity was studied in rabbit articular chondrocytes in primary culture, where cells are in a well-differentiated state as assessed by collagen-type synthesis, as well as in subculture and in retinoic acid-treated cells, where cells are in a dedifferentiated state. Results showed that two different TGases activities are expressed in chondrocytes. One, down-regulated upon retinoic acid treatment of cells, preferentially membrane bound and strongly activated upon trypsin treatment of cell lysates, is expressed at a high level in primary culture. The other one is up-regulated upon retinoic acid treatment, preferentially cytosolic and inactivated upon trypsin treatment of cell lysates. The rate of expression of the TGase down-regulated by RA seems to correlate with the differentiation state of the chondrocyte. This suggests that this TGase activity may have a physiological role in cartilage and merits further study.
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PMID:Transglutaminase activity in rabbit articular chondrocytes in culture. 774 82

We studied the interaction of [125I]fibronectin with human umbilical vein endothelial cells. Endothelial cell monolayers cross-linked [125I]fibronectin which had been preadsorbed to gelatin-coated dishes. The cross-linking of the substrate-immobilized [125I]fibronectin was mediated by cell-associated tissue transglutaminase and occurred more rapidly during the first 30 min after endothelial cell seeding but also continued for several hours after the cells were fully spread. The processing of the [125I]fibronectin was associated with the basolateral surface of the endothelial cell, as demonstrated by the finding that cross-linking did not occur when [125I]fibronectin was presented to the apical surface of confluent monolayers. Transglutaminase activity was not necessary for attachment and spreading of HUVEC on a fibronectin/gelatin matrix. The presence of a nonpeptidyl transglutaminase inactivator rendered the cells more susceptible to detachment by trypsin and destabilized the association of fibronectin with the subendothelial extracellular matrix. Thus, endothelial cells process fibronectin into cross-linked multimers due to the expression of tissue transglutaminase at the basal surface of the cell. This process may serve to stabilize the extracellular matrix and to firmly anchor the cells to the basement membrane.
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PMID:Transglutaminase-mediated processing of fibronectin by endothelial cell monolayers. 790 54

Plasminogen activator inhibitor type 2 (PAI-2) prevents fibrinolysis by blocking plasminogen activators. It is expressed principally by trophoblast cells and macrophages. PAI-2 in trophoblast membranes has been found cross-linked to large complexes apparently catalyzed by trophoblast transglutaminase (Jensen, P. H., Lorand, L., Ebbesen, P., and Gliemann, J. (1993) Eur. J. Biochem. 214, 141-146). Recombinant human PAI-2 was labeled with [14C]putrescine catalyzed by guinea pig liver transglutaminase. The [14C]putrescine-labeled PAI-2 was digested with cyanogen bromide and trypsin, and the peptides were purified by reverse-phase high performance chromatography. Amino acid sequencing and plasma desorption mass spectrometry of the labeled peptides revealed [14C]putrescine incorporation at Gln83, Gln84, and Gln86. These residues are present in a PAI-2-specific region of 33 amino acids that is inserted between helices C and D and which probably represents a unique solvent-exposed domain. A PAI-2 mutant lacking this insertion was determined not to be a substrate for transglutaminase by [14C]putrescine incorporation and could not form transglutaminase-catalyzed polymers. Thus, the unique PAI-2 insertion represents a functional domain that, by virtue of its transglutaminase acceptor sites, allows participation in binding reactions without affecting the inhibitory function of PAI-2.
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PMID:A unique interhelical insertion in plasminogen activator inhibitor-2 contains three glutamines, Gln83, Gln84, Gln86, essential for transglutaminase-mediated cross-linking. 791 Aug 24

The binding, internalization, and proliferation of Ehrlichia risticii in P388D1 cells and equine polymorphonuclear (PMN) leukocytes were studied by immunofluorescent staining and flow cytometric analysis. The binding of ehrlichiae to P388D1 cells at 4 degrees C was dose dependent, and the antigens of bound organisms were susceptible to pronase treatment. Additionally, the binding of ehrlichiae to P388D1 cells was diminished when either P388D1 cells or ehrlichiae were treated with 1% paraformaldehyde for 30 min or 0.25% trypsin for 15 min. These results indicate that the ehrlichial ligand and host cell receptor are likely surface proteins. Following incubation at 37 degrees C, bound E. risticii and/or its antigens were removed with pronase and indirect immunofluorescent staining in the presence of saponin was used to examine intracellular ehrlichiae. Our results indicate that E. risticii was internalized into P388D1 cells within 3 h and proliferated by 48 h of incubation. The microfilament-disrupting agent cytochalasin D and the transglutaminase inhibitor monodansylcadaverine were used to differentiate between phagocytosis (sensitive to cytochalasin) and receptor-mediated endocytosis (sensitive to monodansylcadaverine) of E. risticii by P388D1 cells. In concentrations that produced distinctive morphological changes and inhibited phagocytosis of polystyrene latex beads, cytochalasin D did not suppress the infectivity of E. risticii. Binding, internalization, or proliferation of E. risticii was not affected by cytochalasin D. However, monodansylcadaverine inhibited infection of E. risticii in a dose-dependent manner. The agent did not affect the attachment of ehrlichiae to host cells, but it did suppress internalization and proliferation. These results suggest that E. risticii is internalized by receptor-mediated endocytosis and that productive infection by E. risticii does not depend on phagocytosis by the P388D1 cells. Although E. risticii did not bind to the surface of equine PMN leukocytes at 4 degrees C, organisms were taken up by this cell at 37 degrees C. E. risticii, however, failed to survive in equine PMN leukocytes.
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PMID:Characterization of Ehrlichia risticii binding, internalization, and proliferation in host cells by flow cytometry. 835 1

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

Actin labeled at Gln-41 with dansyl ethylenediamine (DED) via transglutaminase reaction was used for monitoring the interaction of myosin subfragment 1 (S1) with the His-40-Gly-42 site in the 38-52 loop on F-actin. Proteolytic digestions of F-actin with subtilisin and trypsin, and acto-S1 ATPase measurements on heat-treated F-actin revealed that the labeling of Gln-41 had a stabilizing effect on subdomain 2 and the actin filaments. DED on Gln-41 had no effect on the values of K(m) and Vmax of the acto-S1 ATPase and the sliding velocities of actin filaments in the in vitro motility assays. This suggests either that S1 does not bind to the 40-42 site on actin or that such binding is not functionally important. The binding of monoclonal antidansyl IgG to DED-F-actin did not affect acto-S1 binding in the absence of nucleotides, indicating that the 40-42 site does not contribute much to rigor acto-S1 binding. Myosin-induced changes in subdomain 2 on actin were manifested through an increase in the fluorescence of DED-F-actin, a decrease in the accessibility of the probe to collisional quenchers, and a partial displacement of antidansyl IgG from actin by S1. It is proposed that these changes in the 38-52 loop on actin originate from S1 binding to other myosin recognition sites on actin.
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PMID:Myosin-induced changes in F-actin: fluorescence probing of subdomain 2 by dansyl ethylenediamine attached to Gln-41. 878

A monoclonal antibody (5A2) recognizing a segment near the C-terminus of the fibrin(ogen) A alpha-chain (A alpha #529-539) was found to inhibit alpha-chain crosslinking catalyzed by coagulation factor XIIIa and by tissue-transglutaminase. The rapid gamma-chain cross-linking by factor XIIIa was not affected by the antibody. Results obtained from direct binding and competitive immunoassay established that the antigenic determinant recognized by 5A2 was included within the CNBr fragment referred to as CNBr X (A alpha #518-584), and that it survived trypsin digestion but was destroyed by treatment with Staph V-8 protease or chymotrypsin. Reverse-phase (C-18) high performance liquid chromatography (HPLC) was employed to obtain a CNBr X tryptic fingerprint, which was subsequently characterized by compositional and NH2-terminal analysis. Assay of the HPLC column effluent revealed a single peak of 5A2 immunoreactivity that coincided with elution of the eleven-residue tryptic peptide, A alpha #529-539. When this isolated peptide and its parent CNBr fragment were employed as solution phase competitors in the 5A2 immunoassay, the relative cross-reactivities (18.3%, peptide: fragment) indicated that a significant proportion of the 5A2 epitope was preserved within the small peptide. This is a region that is released from fibrinogen early in its degradation by plasmin. Thus, the antibody can be used as a probe for intact fibrin(ogen) and C-terminal (A) alpha-chain fragments, in addition to assessing roles of the A alpha-chain C-terminus in cross-linking.
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PMID:Monoclonal antibody directed to a fibrinogen A alpha #529-539 epitope inhibits alpha-chain crosslinking by transglutaminases. 884 68


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