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)

We investigated the effect of divalent metal ions on the proteolytic cleavage and activation of platelet Factor XIII by thrombin and trypsin. In the absence of metal ions (5 mM EDTA), trypsin and thrombin rapidly degraded platelet Factor XIII (80 kDa) to low-molecular-mass peptides (50-19 kDa) with simultaneous loss of transglutaminase activity. Divalent metal ions protected Factor XIII from proteolytic inactivation with an order of efficacy of Ca2+ greater than Zn2+ greater than Mg2+ greater than Mn2+. Calcium (2 mM) increased by 10- to 1000-fold the trypsin and thrombin concentrations required to degrade Factor XIII to a 19-kDa peptide. Factor XIIIa formed by thrombin in the presence of 5 mM EDTA had one-half the specific activity of Factor XIIIa formed in the presence of calcium. Factor XIII was cleaved by trypsin in the presence of 5 mM Ca2+ to a 51 +/- 3-kDa fragment that had 60% of the original Factor XIIIa activity. A similar tryptic peptide formed in the presence of 5 mM EDTA did not have transglutaminase activity. In the presence of 5 mM Mg2+, thrombin cleaved Factor XIII to a major 51 +/- 3-kDa fragment that had 60% of the Factor XIIIa activity. Mn2+ (0.1-5 mM) limited trypsin and thrombin proteolysis. The resulting digest containing a population of Factor XIII fragments (50-14 kDa) expressed 50-60% transglutaminase activity of Factor XIIIa. Factor XIII was fully activated by both trypsin and thrombin in the presence of 5 mM Zn2+, resulting in two fragments of 76 and 72 kDa. We conclude that the binding of divalent metal ions to platelet Factor XIII induces conformational changes in the protein that alter its susceptibility to proteolysis and influence the expression of transglutaminase activity.
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PMID:The binding of divalent metal ions to platelet factor XIII modulates its proteolysis by trypsin and thrombin. 289 89

While the transglutaminase activity is associated exclusively with the thrombin-cleaved a chains of plasma Factor XIII, there is little information regarding the role of the b-chains. The present investigations were undertaken to clarify the role of the b-chains during proteolytic activation of plasma factor XIII a-chains. The a-chains of platelet Factor XIII (a2) were extremely sensitive to alpha-thrombin proteolysis, especially in the presence of 5 mM EDTA, resulting in two major fragments with molecular masses 51 +/- 3 kDa and 19 +/- 4 kDa. Furthermore, fibrin enhanced the alpha-thrombin proteolysis of thrombin-cleaved platelet Factor XIII a-chains in presence of CaCl2 or EDTA, resulting in several peptide fragments with molecular masses from 51 +/- 3 kDa to 14 +/- 4 kDa. By contrast, thrombin-cleaved a-chains of plasma Factor XIII (a2b2) were not further degraded by alpha-thrombin in presence of 5 mM EDTA. Even in the combined presence of 5 mM EDTA and 0.1 mg/ml fibrin, alpha-thrombin proteolysis of plasma Factor XIIIa was limited to the formation of a 76 kDa fragment (= Factor XIIIa), a 51 +/- 3 kDa fragment and trace amounts of a 14 +/- 4 kDa species. Platelet Factor XIII proteolyzed by 500 nM alpha-thrombin in presence of 5 mM EDTA expressed less than 20% of enzymatic activity obtained when platelet Factor XIII was activated in presence of 5 mM CaCl2. In contrast, plasma Factor XIII activated by 500 nM apha-thrombin in presence of 5 mM EDTA expressed nearly 65% of original transglutaminase activity. Likewise, when plasma Factor XIII was proteolyzed by 100-1000 nM gamma-thrombin in presence of 5 mM CaCl2 or 5 mM EDTA, maximal transglutaminase activity was observed. However, when platelet Factor XIII was similarly treated with gamma-thrombin in presence of 5 mM EDTA, only one-half the original transglutaminase activity was obtained. The b-chains thus appear to mimic the function of Ca2+ in preserving transglutaminase activity of thrombin-cleaved a-chains. The b-chains of plasma Factor XIII were not degraded by either alpha- or gamma-thrombin treatment, in presence of 5 mM EDTA or 5 mM CaCl2. Both platelet and plasma Factor XIII a-chains were degraded by trypsin to fragments with molecular masses of 51 +/- 3 kDa and 19 +/- 4 kDa in presence of 5 mM CaCl2 and to fragments with molecular masses of 19 +/- 4 kDa and lower, in presence of 5 mM EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:b-chains prevent the proteolytic inactivation of the a-chains of plasma factor XIII. 290 Dec 75

The interaction of fibrinogen and fibronectin with hepatocytes has been dissociated into distinct binding and cross-linking steps. Binding and cross-linking of 125I-labeled ligands were both decreased by transglutaminase inhibitors, but not by heparin or hirudin. Transglutaminase activity was manifest by Ca2+-dependent incorporation of [14C]putrescine into cells. Preferential cross-linking of fibrinogen A alpha over gamma chains, and lack of inhibition by heparin or hirudin indicates the involvement of tissue transglutaminase, and not Factor XIIIa. Hepatic transglutaminase activity, as well as binding and cross-linking of fibrinogen and fibronectin, were maximally supported by Ca2+, partially supported by Mn2+ and Sr2+, and markedly decreased by Mg2+ and Ba2+. In contrast, Co2+ supported binding but not cross-linking or transglutaminase activity, indicating that binding and cross-linking are dissociable events. This conclusion was corroborated by the finding that fibrinogen fragments D95 and D78 both inhibited Ca2+-dependent fibrinogen binding without being cross-linked themselves. Ligand binding in the presence of either cation was localized to the cell surface as evidenced by its trypsin sensitivity. Thus, fibrinogen and fibronectin binding to hepatocytes is independent of transglutaminase activity, whereas cross-linking of these adhesive macromolecules requires an enzymatically active cellular transglutaminase. In addition, fibrinogen binding appears to be mediated by molecular determinants present in fragment D78.
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PMID:Dissociation of fibrinogen and fibronectin binding from transglutaminase-mediated cross-linking at the hepatocyte surface. 290 77

Purified platelet Factor XIII was radioiodinated and then partially degraded by thrombin or trypsin, and a fibrin-binding fragment was identified by autoradiography and immunoblotting following separation by SDS/polyacrylamide-gel electrophoresis. Limited proteolysis of 125I-Factor XIII by thrombin or trypsin produced an 125I-51 kDa fragment and an unlabelled 19 kDa fragment. The 51 kDa fragment was purified by h.p.l.c. on a TSK-125 gel-filtration column. Partial amino acid sequence analysis of the 51 kDa fragment indicated that it was similar in sequence to the Gly38-Lys513 segment in placental Factor XIII a-chain. More than 70% of the 51 kDa fragment bound to fibrin, whereas the 19 kDa fragment did not bind. The active site was localized to the 51 kDa fragment since this fragment expressed transglutaminase activity, cross-linked fibrin and fibrinogen and incorporated iodo[14C]acetamide into the active-site cysteine residue. Isolation of a fibrin-binding fragment expressing transglutaminase activity demonstrates that each a-chain of the dimeric Factor XIIIa could function independently to cross-link fibrin. The fibrin-binding site could play an important role in localizing Factor XIIIa to the fibrin clot.
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PMID:Isolation of a fibrin-binding fragment from blood coagulation factor XIII capable of cross-linking fibrin(ogen). 306 50

A marked decrease in activity of ornithine decarboxylase in thymus and spleen occurs soon after treatment of rats with a glucocorticoid. In the present study, evidence was obtained that extracts of these tissues prepared 5 h after administration of dexamethasone, when the enzyme activity is very low, contain an inhibitor of ornithine decarboxylase. The inhibitor is also present at 12 h after treatment and, in lesser amount, at 2.5 h, but was not evident at 24 h. The inhibitory activity was destroyed by treatment with heat or with trypsin, and was not lost on dialysis of the extract. Preliminary experiments indicate that the Mr of the inhibitor is greater than 50 000, which differentiates it from antizyme, an inhibitor of ornithine decarboxylase found in several other cell types. The inhibitor seems to act by a non-catalytic and non-competitive mechanism. The inhibition is dependent on the amount of inhibitor and does not change with time. Since inhibition is not changed by dialysis of the inhibitory extract, its activity apparently does not require small-Mr substances. This differentiates it from inhibitors which inactivate ornithine decarboxylase by covalent modification, such as the polyamine-dependent protein kinase or transglutaminase. The formation of this inhibitor is an early event in lymphoid tissues in response to dexamethasone and may be important in causing the inhibition of cell division which precedes the destruction of lymphocytes.
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PMID:An inhibitor of ornithine decarboxylase in the thymus and spleen of dexamethasone-treated rats. 397 59

We have investigated the structural and functional differences between chicken and human cellular fibronectin by comparing the tryptic peptide patterns using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by analyzing the binding properties of isolated trypsin-resistant polypeptide fragments. Although the overall functional organization of chicken and human cellular fibronectins was similar, the tryptic patterns obtained from these two molecules were strikingly different. For example, the tryptic digest of chicken cellular fibronectin contained two unique peptide fragments having molecular sizes of 45 and 70 kilodaltons. The previously unidentified carboxyl-terminal 45-kDa fragment is an intermediate that appears between 15 to 120 s of digestion. The 70-kDa fragment binds to gelatin, to fibrin (with unusually high apparent affinity), to heparin (at low ionic strength), and to fixed Staphylococcus aureus cells; it also contains an acceptor site for factor XIIIa (plasma transglutaminase). These results suggest that the functional domains of chicken and human fibronectins remain constant and that structural variations occur in the protease-susceptible regions of the molecule. The present findings are discussed in terms of the previously existing discrepancies in the literature on fibronectin.
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PMID:Structural and functional comparisons of chicken and human cellular fibronectins. 614 22

The membranous fraction isolated from stratum corneum by 8M urea-beta ME containing alkaline buffer (pH 9.0) is quite crude when observed by electron microscopy. However, this procedure may be useful for clinical samples, as one can isolate and compare both the soluble (interfilamentous) fraction and the keratin filament from the same sample in addition to the residues (membranous fraction). A further purified membranous fraction was isolated by a new method. Human stratum corneum was chopped and treated with 8M urea-50 mM Tris-HCl (pH 9.0), digested by the use of trypsin, and the product fractionated by a sucrose density gradient to obtain separate single cells without the cytoplasm. One sample was then treated with trypsin for 1 hour and another with urea buffer for 24 hours. Observations revealed a thickened inner membrane (marginal band) of approximately 150A. Each of the membranous samples contained a level of half-cystine markedly higher in amount (around 100/1,000) and involved mostly in the epsilon-(gamma-glutamyl) lysine cross-linkages (around 30%). In order to compare the membranous fraction of horny and living cells (marginal bands and plasma membranes), the fraction was then isolated from living cells. The relative amino acid composition of the membranous fraction of the plasma membrane resembled that of human erythrocytes, but was quite different from that of the marginal band. These comparative studies of biochemical and morphological features suggested the importance of S-S cross-linking enzymes and transglutaminase in the transformation mechanism of the marginal band.
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PMID:Comparative studies of the marginal band and plasma membrane of the epidermis. 619 46

We have recently demonstrated that exposure of rat myoblasts to anti-rat myoblast antiserum results in two- to three-fold activation of hexose transport. The present communication reports the possible mechanism(s) by which specific antibody can bring about such activation. Studies with Fab and Fc fragments indicate that the binding of Fab to specific cell surface component(s) is not sufficient to trigger activation of hexose transport; the immunoglobulin G (IgG) mediated dimerization of membrane components is required for this process. Although cytochalasin D has no effect on hexose transport in control and antibody-treated cells, pretreatment of cells with this inhibitor prevents antibody-mediated activation of hexose transport. It may be inferred from this observation that proper disposition of membrane components is required for the dimerization of membrane receptors. Since this activation of hexose transport is an irreversible process, it is possible that covalent modification of membrane components may have occurred as a result of antibody treatment. Pretreatment of cells with ammonium chloride or methylamine is found to abolish the antibody-mediated activation of hexose transport, even though these inhibitors have no effect on hexose transport in control and antibody-treated cells. These inhibitors may be acting on transglutaminase and (or) on some other proteins involved in the activation process. Several lines of evidence suggest that limited proteolytic cleavage of membrane components may be involved in the antibody-mediated activation of hexose transport. First, pretreatment with several protease inhibitors prevents activation of hexose transport. Second, several cell surface proteins are missing in antibody-treated cells. Third, limited proteolysis of cell surface proteins with trypsin can also bring about activation of hexose transport. In view of the fact that proteolytic activity cannot be detected in various IgG and serum preparations, it seems likely that endogenous membrane associated proteases may be involved in this activation process.
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PMID:Mechanism of antibody stimulation of hexose transport in rat myoblasts. 637 38

The complete amino acid sequence of the NH2-terminal domain obtained after trypsin digestion of human plasma fibronectin has been determined. It contains residues 1-259 and has a Mr of 29,000. The 29-kDa fragment was isolated from the other major tryptic cleavage products of 200, 180, and 31 kDa by affinity chromatography on gelatin- and heparin-Sepharose columns. The two high Mr fragments bound to gelatin and were easily removed; the 31-kDa fragment failed to bind to heparin while the 29-kDa fragment did and was eluted with 0.15 M NaCl. The 29-kDa domain has a blocked NH2-terminal (pyrrolidone carboxylic acid) which was removed by digestion with pyroglutamate aminopeptidase, and the amino acid sequence of the first 36 residues was obtained. The sequence showed a glutamine residue at position 3 which is probably the acceptor site for transglutaminase as reported for bovine fibronectin. Extensive trypsin digestion of the completely reduced and alkylated 29-kDa fragment yielded twenty-four peptides which were separated and purified by high performance liquid chromatography; their amino acid composition and amino acid sequence has been determined and the arrangement of peptides was achieved by comparison with the sequence recently reported for bovine fibronectin. The sequences in human and bovine fibronectin were nearly identical with only nine amino acid differences which can all be explained by single base substitutions. Apparently this domain is highly conserved in the two species studied thus far.
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PMID:Primary structure of human plasma fibronectin. The 29,000-dalton NH2-terminal domain. 663 Feb 2

The mechanism of insulin's action upon intracellular proteolysis in isolated hepatocytes was studied. At 37 degrees C insulin inhibited intracellular degradation of intracellular proteins in a dose-dependent manner. A maximal 40% inhibition of intracellular proteolysis was achieved at an insulin concentration of 500 ng/ml with a half-maximal inhibition observed at 2.5 ng/ml of insulin. Insulin inhibited intracellular proteolysis both in the presence and in the absence of amino acids in the incubation mixture. Low concentrations of trypsin (10 micrograms/ml) mimicked insulin's effect upon glucose incorporation into glycogen, but not on intracellular proteolysis. Four protease inhibitors (phenylmethylsulfonyl fluoride (0.5 mM), p-nitrophenyl-p-guanidinobenzoate (0.25 mM), p-tosyl-L-arginine methyl ester (1 mM), and N alpha-p-tosyl-L-lysine chloromethyl ketone (1 mM) blocked the stimulatory effect of insulin upon [14C]glucose incorporation into glycogen, but did not affect the inhibitory action of insulin upon intracellular proteolysis. These results suggest that the mechanism of insulin's action upon intracellular proteolysis differs from that involved in stimulation of glycogenesis. Low temperature (15 degrees C) and short time exposure (10 min) of the hepatocytes to insulin eliminated the inhibitory effect of insulin on intracellular proteolysis. Similarly, insulin's effect on intracellular proteolysis was eliminated by dansylcadaverine, a transglutaminase inhibitor that blocked insulin internalization. In contrast, dansylcadaverine had no effect on insulin's ability to stimulate [14C]glucose incorporation into glycogen. These experiments strongly suggest the necessity of insulin internalization for its inhibitory effect on endogenous protein degradation.
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PMID:Inhibition of intracellular proteolysis by insulin in isolated rat hepatocytes. Possible role of internalized hormone. 674 50


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