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

Thrombin converts fibrinogen to fibrin monomer by cleaving fibrinopeptides A and B (FPA and FPB) from the amino terminal ends of the A (alpha) and B (beta) chains. A radioimmunoassay capable of measuring the A peptide in human blood as an index of thrombin action in vivo has been described previously. This paper describes the development of a radioimmunoassay for FPB and the use of both assays in the demonstration of distinctive patterns of cleavage of the amino terminal ends of the A (alha) and B (beta) chains of fibrinogen by various enzymes. Antisera were raised in rabbits to a synthetic analogue of FPB coupled to bovine serum albumin. FPB analogue was couple to desaminotyrosine and radiolabeled with 125I by the chloramine-T technique. The radiolabeled peptide was bound by the antiserum, and binding was inhibited by synthetic or native FPB. Unbound tracer was separated from bound tracer by charcoal adsorption. The senistivity of the assay was such that 50% inhibition of binding of the tracer was caused by 1.25 ng of the native FPB. Fibrinogen was treated with thrombin, plasmin, trypsin, Reptilase, and an extract of the venom from Ancistrodon contortrix contortrix (ACC). After ethanol precipitation and centrifugation, dialysates of enzymatically altered fibrinogen were assayed for FPA and FPB. The action of thrombin on fibrinogen resulted in a rapid release of FPA and a slower release of FPB. Plasmin cleaved a segment(s) of the B (beta) chain which included FPB but cleaved no detectable FPA-containing material for the first 2 h of incubation. In the case of plasmin-treated fibrinogen, the dialysates had been further treated with thrombin before being assayed for FPA and FPB. Trypsin rapidly cleaved both peptides, the B before the A. Reptilase cleaved only FPA in 24 h. ACC cleaved FPB at a rapid rate, with a slowere cleavage of FPA. The distinctive cleavage patterns produced by the serine proteases may be useful in interpreting the levels of FPA and FPB measured in human blood and in studying the generation of FPA and FPB in clinical blood samples.
 ...
PMID:Radioimmunoassay of human fibrinopeptide B and kinetics of fibrinopeptide cleavage by different enzymes. 5 Mar 28

A series of substituted benzamidines has been examined for their inhibitory activity against the human serine proteases--trypsin, thrombin, plasmin, and C1s, a subunit of the first component of complement. The inhibition constants obtained for each enzyme were correlated with physical-chemical properties of the substituent group using the quantitative structure-activity relationship approach. This analysis indicated that plasmin and C1s are very similar in their interactions with substituted benzamidines. The binding of benzamidines in both enzymes was affected by electron donation from the substituent and its hydrophobicity. Thrombin-benzamidine interaction was affected only by the hydrophobicity of the substituent. Trypsin displayed a complex interaction with substituted benzamidines, and interaction was dependent on molar refractivity and molecular weight. Certain substituents deviated significantly from the interactions predicted by the analysis. These compounds, the (m- and p-amidinophenyl)pyruvic acids, when analyzed by computer modeling, suggested that direct interaction between the substituent and the enzyme surface is important in assessing the effect of substituent groups on inhibitory activity.
 ...
PMID:Inhibition of four human serine proteases by substituted benzamidines. 15 12

Thrombin stimulates cell proliferation in cultures of normal chick embryo fibroblasts but not in cells transformed with Rous sarcoma virus. Analysis of medium conditioned by Rous-sarcoma-virus-transformed cultures demonstrates that these cells do not secrete molecules that can inhibit or inactivate thrombin. The interaction of thrombin with these cells was investigated with enzymatically active 125I-thrombin. The amount of cell-associated 125I-thrombin was found to be three times greater with normal cells than with transformed cells. In both types of cell, greater than 50% of the total cell-associated 125I-thrombin was found as a component that was not dissociated from the cells by trypsin treatment, an observation suggesting that a significant portion was not on the cell surface. The amount of the trypsin-insensitive fraction increases with time up to 12 hr, whereas the trypsin-sensitive fraction is saturated after 1-4 hr. Autoradiography of thin sections of 125I-thrombin-treated cells observed by electron microscopy reveals that after 10 hr incubation greater than 70% of the label is localized in the cytoplasm of both normal and transformed cells. Autoradiograms of sodium dodecyl sulfate/polyacrylamide slab gels demonstrate that 40% of the intracellular label is the size of native thrombin with the remainder in two large fragments of 22,000 and 19,500 daltons.
 ...
PMID:Binding and internalization of thrombin by normal and transformed chick cells. 19 17

Perfusion of thrombin and trypsin solutions through the frog carotid labyrinth acts on the carotid chemoreceptors and evokes reflex response of the anticoagulating system. The similarity of effects of both these agents seems to be due to similarity of their structures. Other agents acting on the frog vascular chemoreceptors: sodium chloride hypoxia, lobeline,--cause no activation of the reflex anticoagulating system. Thrombin is concluded to be the adequate and specific irritant of the vascular chemoreceptors of the frog anticoagulating system.
 ...
PMID:[Specificity of the effect of thrombin on the chemoreceptors of the frog carotid labyrinth]. 30 Nov 3

Thrombin-mediated platelet aggregation and release is enhanced by the presence of C3, C5, C6, C7, C8, and C9 of human complement. The interaction of thrombin with its receptor on the platelet membrane initiates activation of complement on the platelet surface. Trypsin-mediated platelet function is not enhanced by the addition of complement, probably because trypsin has no receptor on the platelet surface so activation of complement is triggered in the fluid phase and not on the platelet surface. Activation of complement by thrombin led to production of dimers of the C5b-9 complex on the platelet surface. These complexes were eluted from the platelet membrane and were identified physicochemically and morphologically. The mechanism of complement-induced enhancement of platelet function is not clear, however, it probably is mediated via the arachidonic acid transormation pathway because this activity was blocked by known inhibitors of cyclo-oxygenase, namely, aspirin and indomethacin.
 ...
PMID:Human complement in thrombin-mediated platelet function: uptake of the C5b-9 complex. 47 64

Thrombin first activates and then inactivates factor VIII and for this reason thrombin has been considered responsible for the inactivation of factor VIII which occurs during clotting. Experiments described in this paper indicated that the activity of factor VIII is not reduced in factor IX or factor X deficient sera, while on the other hand this factor becomes inactivated in blood anticoagulated with high concentrations of hirudin which inhibit thrombin activity completely. This suggests that some other factor, besides thrombin, which is generated only in trace amounts in factor IX or factor X deficient plasmas, is also able to inactivate factor VIII. Purified factor X activated with insolubilized trypsin was added to purified preparations of factor VIII, which were free of both fibrinogen and prothrombin. Factor X a was allowed to act for 5-60 minutes and then inactivated with phenylmethanesulfonyl fluoride. Depending on the duration of the action of factor X a partial or complete inactivation of factor VIII was observed. This inactivation was also observed in the presence of hirudin, thus excluding the possibility that the effect was due to contamination with trace amounts of thrombin.
 ...
PMID:Inactivation of factor VIII by a mechanism independent of the generation of thrombin. 50 1

The effect of thrombin on the phosphorylating activity of platelet membranes was compared to that of trypsin. Preincubation of non-32P phosphorylated platelet membranes with or without either of these two enzymes resulted in a considerable loss of membrane protein kinase activity which was most severe when trypsin was used. Protein kinase activity and endogenous protein acceptors decreased in parallel. 32P-phosphorylated membranes showed a slow but progressive loss of label which was accelerated by trypsin. Thrombin under these conditions prevented the loss of 32P-phosphate. These results are interpreted to indicate a thrombin-induced destruction of a phosphoprotein phosphatase. The protein kinase activity of phosphorylated platelet membranes using endogenous or exogenous protein substrates showed a significant reduction compared to non-phosphorylated membranes suggesting a deactivation of protein kinase by phosphorylation of platelet membranes. Neither thrombin nor trypsin caused a qualitative change in the membrane polypeptides accepting 32P-phosphate but resulted in quantitative alterations of their ability to become phosphorylated.
 ...
PMID:Effect of thrombin on phosphorylation of platelet membrane proteins. 98 70

This communication explores the capacity of different proteases to stimulate DNA synthesis in resting chick embryo fibroblasts and to cause the removal of cell membrane proteins previosly postulated as important in the regulation of growth and division of cells. Thrombin, a highly specific protease and a known mitogen, was incubated with chick embryo fibroblasts, and analysis was made of the cell membrane proteins. Of particular interest were a protein of molecular weight 250,000, which is known to be readily removed by the action of trypsin and is not present in most transformed cells, and two other proteins, which are reduced in amount in transformed as compared to confluent resting cell cultures. None of these three proteins was removed by thrombin when the latter was added to confluent cells in concentrations sufficient to cause significant increase in DNA synthesis twelve hours after stimulation by the protease. The presence or absence of these proteins in the membranes of confluent resting or transformed cells of chick embryo fibroblasts does not seem to be directly related to the process of regulation of DNA synthesis and cellular division.
 ...
PMID:The role of surface proteins in cell proliferation as studied with thrombin and other proteases. 105 23

The interaction of bovine thrombin [EC 3.4.21.5] with synthetic substrates and products was studied. The enzyme was purified from Parke-Davis topical thrombin. The purification process afforded some preparations with different clottin specific activities but with similar esterase specific activities. The preparation having highest clotting specific activity and that having lowest clotting activity were tentatively named thrombin-C and thrombin-E, respectively. Kinetic parameters for the hydrolysis of synthetic substrates and normality titrants were determined on the basis of active enzyme quantity, which was assayed by means of a fluorometric normality titrant. It was shown that thrombin-E was acylated by the substrates more slowly than thrombin-C, while deacylation proceeded at similar rates in the two preparations. The results were also compared with those obtained with bovine trypsin [EC 3.4.21.4]. The acylation rates of both thrombin preparations were markedly lower than that of trypsin, while the deacylation rates of the former were only slightly lower than that of the latter. The effects of various product-type inhibitors, such as benzyloxycarbonyl-, benzoyl-, and tosyl-L-arginine, were also examined. Thrombin was affected by these inhibitors not competitively, though trypsin was inhibited competitively.
 ...
PMID:Comparison of the catalytic properties of thrombin and trypsin by kinetic analysis on the basis of active enzyme concentration. 124 85

Thrombin is a multifunctional serine proteinase that plays a key role in coagulation while exhibiting several other key cellular bioregulatory functions. The X-ray crystal structure of human alpha-thrombin was determined in its complex with the specific thrombin inhibitor D-Phe-Pro-Arg chloromethylketone (PPACK) using Patterson search methods and a search model derived from trypsinlike proteinases of known spatial structure (Bode, W., Mayr, I., Baumann, U., Huber, R., Stone, S.R., & Hofsteenge, J., 1989, EMBO J. 8, 3467-3475). The crystallographic refinement of the PPACK-thrombin model has now been completed at an R value of 0.156 (8 to 1.92 A); in particular, the amino- and the carboxy-termini of the thrombin A-chain are now defined and all side-chain atoms localized; only proline 37 was found to be in a cis-peptidyl conformation. The thrombin B-chain exhibits the characteristic polypeptide fold of trypsinlike serine proteinases; 195 residues occupy topologically equivalent positions with residues in bovine trypsin and 190 with those in bovine chymotrypsin with a root-mean-square (r.m.s.) deviation of 0.8 A for their alpha-carbon atoms. Most of the inserted residues constitute novel surface loops. A chymotrypsinogen numbering is suggested for thrombin based on the topological equivalences. The thrombin A-chain is arranged in a boomeranglike shape against the B-chain globule opposite to the active site; it resembles somewhat the propeptide of chymotrypsin(ogen) and is similarly not involved in substrate and inhibitor binding. Thrombin possesses an exceptionally large proportion of charged residues. The negatively and positively charged residues are not distributed uniformly over the whole molecule, but are clustered to form a sandwichlike electrostatic potential; in particular, two extended patches of mainly positively charged residues occur close to the carboxy-terminal B-chain helix (forming the presumed heparin-binding site) and on the surface of loop segment 70-80 (the fibrin[ogen] secondary binding exosite), respectively; the negatively charged residues are more clustered in the ringlike region between both poles, particularly around the active site. Several of the charged residues are involved in salt bridges; most are on the surface, but 10 charged protein groups form completely buried salt bridges and clusters. These electrostatic interactions play a particularly important role in the intrachain stabilization of the A-chain, in the coherence between the A- and the B-chain, and in the surface structure of the fibrin(ogen) secondary binding exosite (loop segment 67-80).(ABSTRACT TRUNCATED AT 400 WORDS)
 ...
PMID:The refined 1.9-A X-ray crystal structure of D-Phe-Pro-Arg chloromethylketone-inhibited human alpha-thrombin: structure analysis, overall structure, electrostatic properties, detailed active-site geometry, and structure-function relationships. 130 49


1 2 3 4 5 6 7 8 9 10 Next >>