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)

Z-D-Phe-Pro-boroMpg-OPin (9a)1,2 has been shown previously to be a highly specific inhibitor of thrombin in spite of lacking an arginine-like guanidino group at the P1 site. A range of compounds have been synthesized based upon this lead compound, varying the neutral side chain at the P1 site. Of the 20 examples based upon the structures at P2 and P3 of Z-D-X-Pro (X being Phe or beta,beta-diphenylalanine), all were found to be effective inhibitors of thrombin (Ki's between 10 and 100 nM). Furthermore all exhibited a high specificity toward thrombin having values for a Ki(trypsin)/Ki(thrombin) ratio of between 10- and 100-fold. High ratio values were found for a number of the compounds tested against a range of serine proteinases (plasmin, factor Xa, kallikrein, urokinase, protein Ca, chymotrypsin, elastase, and cathepsin G). As far as potency toward thrombin, compounds containing the methoxypropyl group at P1 were favored over those with a methoxy grouping on a shorter alkyl chain (8) or without the methoxy group (1-5). The compounds display potent anticoagulant activity with values for 18 in thrombin time of 0.63 microM and in activated partial thromboplastin time of 2.0 microM. 11B NMR has been used to confirm interaction of the boron atom with the active site. From the high specificity shown with all the compounds we propose that the compounds, constitute a new class of thrombin inhibitors.
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PMID:Characterization of a class of peptide boronates with neutral P1 side chains as highly selective inhibitors of thrombin. 773 10

From the saliva of the vampire bat Desmodus rotundus, we isolated an unknown anticoagulant protein which we have named draculin. Its molecular mass as determined by non-reduced SDS-PAGE is about 83 kDa. The reduced polypeptide shows a slower migration. HPLC in a molecular sieve matrix yields a single, symmetrical peak corresponding to 88.5 kDa. Isoelectric focusing shows an acidic protein with pI = 4.1-4.2. Aminoacid analysis is compatible with a single chain polypeptide of about 80 kDa. Cyanogen bromide cleavage yields a single 16-aminoacid peptide, corresponding to the amino-terminus of the native molecule. Draculin inhibits the activated form of coagulation factors IX and X. It does not act on thrombin, trypsin, chymotrypsin and does not express fibrinolytic activity. The inhibition is immediate and not readily reversible, with a stoichiometry of about two molecules of draculin per molecule of factor IXa or Xa. Surprisingly, the inhibitory activity against either factor is not affected by the presence of the other. Draculin binds quantitatively to either immobilised factor Xa or factor IXa. Our preliminary interpretation is that there are two forms of draculin that hardly differ in structure. Both bind to factor Xa and to factor IXa but one form inhibits factor Xa and the other inhibits factor IXa. When added to plasma, draculin increases the lag phase as well as the height of the peak of thrombin generation.
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PMID:Purification and partial characterization of draculin, the anticoagulant factor present in the saliva of vampire bats (Desmodus rotundus). 774 May 3

Ecotin, an Escherichia coli periplasmic protein of 142 amino acids, has been shown to be a potent inhibitor of a group of homologous serine proteases with widely differing substrate recognition. It is highly effective against a number of enzymes, including both pancreatic and neutrophil-derived elastases, chymotrypsin, trypsin, factor Xa, and kallikrein. Recent structural and functional studies on ecotin and its interactions with different serine proteases have clarified these initial observations and revealed the remarkable features of this protein in inhibiting a strikingly large subset of the chymotrypsin family of serine proteases. The structures of the ecotin:serine protease complexes provide the first examples of protein-protein recognition where the concept of specificity of interactions needs to be reexamined. The binding sites show a fluidity of protein contacts derived from ecotin's innate flexibility in fitting itself to proteases while strongly interfering with their function.
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PMID:Ecotin: lessons on survival in a protease-filled world. 775 4

Prothrombin is a vitamin K-dependent plasma protein composed of several functional domains, which is proteolytically activated into thrombin by factor Xa in the presence of factor Va, Ca2+, and phospholipids. During the activation, prothrombin is cleaved into three fragments: fragment 1, containing a domain rich in gamma-carboxyglutamic acid residues and kringle 1 domain; fragment 2, containing the kringle 2 domain; and a protease catalytic domain, thrombin. Here we studied the interaction site for factor Xa in human prothrombin during the activation. The isolated fragment 2 inhibited the activation of prothrombin by either prothrombinase complex or factor Xa alone in a dose-dependent manner, whereas fragment 1 and diisopropylphosphate (DIP)-thrombin did not. Factor Xa directly bound to fragment 2 immobilized to microwell plates with a Kd of 9.0 x 10(-8) M, but not to fragment 1 or DIP-thrombin. Factor Xa also bound to immobilized prothrombin and prethrombin 1 with Kds of 2.0 x 10(-7) and 1.5 x 10(-7) M, respectively, suggesting that factor Xa interacts with the kringle 2 domain in these molecules. The binding of factor Xa to immobilized fragment 2 was Ca(2+)-dependent with an optimal concentration at 6 mM. In the presence of Ca2+, the interaction was enhanced by phospholipids in a concentration-dependent manner. To localize the factor Xa-binding site in the kringle 2 domain, fragment 2 was digested with lysyl endopeptidase and then trypsin after reduction and S-carboxymethylation. The resulting peptides were immobilized to microwell plates and assayed for factor Xa binding ability. The amino acid sequence of the peptide positive in the assay was determined to be residues His205 to Arg220. Factor Xa bound to a synthetic peptide corresponding to the residues His205 to Arg220 immobilized to microwell plates. The peptide inhibited factor Xa-catalyzed activation of prothrombin, but a peptide with the reversed sequence of His205 to Arg220 did not. These findings indicate that factor Xa interacts with at least a linear sequence, His205 to Arg220, in the kringle 2 domain of prothrombin during its activation into thrombin.
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PMID:Blood coagulation factor Xa interacts with a linear sequence of the kringle 2 domain of prothrombin. 785 76

A DNA construct encoding human big endothelin (Big ET) preceded by the factor Xa protease recognition site (Ile-Glu-Gly-Arg), fused in frame to the maltose binding protein sequence, has been introduced in DH5-alpha cells. The fusion product (MBP-Big ET) was expressed at a concentration close to 100 micrograms/ml of culture broth and constituted approximately 50% of the total protein content. Crude cell extracts containing the fusion product have been directly treated with trypsin under mild denaturing conditions in order to release big endothelin (1-37) from the adduct. Cleavage yield of the MBP-Big ET adduct was close to 70%. Big ET(1-37) was separated from unrelated peptides derived from the tryptic digest of the bacterial extract by affinity chromatography. The affinity column was prepared by immobilizing a protease resistant peptide ligand able to recognize Big ET with sufficient affinity, selectivity, and specificity. From the affinity step (recovery, 90%), recombinant Big ET(1-37) was obtained with a purity close to 80%. The affinity-purified recombinant product was then digested with alpha-chymotrypsin in order to release endothelin (1-21), which was then purified by RP-HPLC. With this two-step purification protocol, 3 micrograms of endothelin was recovered from 1 ml of bacterial broth, with a purity close to 95%.
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PMID:High yield expression and purification of human endothelin-1. 785 25

Novel thrombin inhibitors, bacithrocins A, B and C, have been isolated from the culture broth of Bacillus laterosporus Laubach NR 2988. The structures of these inhibitors have been determined to be N-acyl-L-phenylalanyl-DL-arginal by the 2D-NMR experiments on their oxidation products and by amino acid analysis. Bacithrocin A inhibits thrombin, factor Xa and trypsin with IC50s of 48, 13 and 0.65 microM, respectively, which are similar to those of bacithrocins B and C. Bacithrocins prolong the clotting time induced by thrombin and factor Xa.
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PMID:Bacithrocins A, B and C, novel thrombin inhibitors. 792 97

DX-9065a is an orally active newly synthesized and specific inhibitor for factor Xa. We have examined the property of DX-9065a in vitro and ex vivo. DX-9065a prolonged human plasma recalcification time, APTT and PT. Its doubling concentrations for clotting times of each coagulation assay were 0.49, 0.97 and 0.52 microM, respectively. Kinetic study revealed that DX-9065a inhibited competitively human factor Xa (Ki value: 41 nM). Ki values (microM) for other human serine proteases were as follows; thrombin > 2000, trypsin 0.62, chymotrypsin > 2000, plasmin 23, t-PA 21, plasma kallikrein 2.3 and tissue kallikrein 1000. DX-9065a up to 100 microM had no effects on human platelet aggregation. After intravenous or oral administration, DX-9065a significantly prolonged APTT and PT with a dose dependent manner. These effects were well correlated with anti-Xa activity in plasma. These results suggest that DX-9065a may become an anticoagulant by means of the specific inhibition of factor Xa.
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PMID:DX-9065a, a new synthetic, potent anticoagulant and selective inhibitor for factor Xa. 802 95

Antistasin, a potent inhibitor of the blood coagulation factor Xa, is the prototype of a novel family of serine-proteinase inhibitors. We have now isolated, sequenced and characterized an antistasin-type inhibitor from the medical leech Hirudo medicinalis. Hirustasin (Hirudo antistasin) was purified to apparent homogeneity by cation-exchange and affinity chromatography. Amino acid sequencing of the 55 amino acid protein (M(r) 5866) revealed that hirustasin is the only antistasin-type protein known to consist of one domain only; 27% and 32% sequence identity was found to the first and second domains of antistasin, respectively, and a nearly exact conservation of the spacing of the ten cysteine residues. Hirustasin is the first inhibitor of tissue kallikrein identified in leeches, and is also a tight-binding inhibitor of trypsin, chymotrypsin and neutrophil cathepsin G. However, despite the high similarity to antistasin, particularly in the vicinity of the putative reactive-site peptide bond, hirustasin neither inhibits blood coagulation in vitro nor amidolytic activity of isolated factor Xa. Thus, structural elements other than the reactive site sequence significantly influence the specificity of antistasin-type proteinase inhibitors.
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PMID:Isolation and characterization of hirustasin, an antistasin-type serine-proteinase inhibitor from the medical leech Hirudo medicinalis. 811 45

The full-length cDNA encoding a novel human intracellular serine proteinase inhibitor has been sequenced and found to encode a 376 amino acid protein (M(r) approximately 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the cytoplasmic antiproteinase has the majority of structural motifs conserved among the greater superfamily of serine proteinase inhibitors, or serpins. On the basis of several criteria such as amino acid identity and the absence of a classical N-terminal signal peptide, the cytoplasmic antiproteinase represents a new member of the intracellular serpin family. Further inspection of the cytoplasmic antiproteinase amino acid sequence identified three potential N-glycosylation sites and Arg341-Cys342 as the reactive site P1-P1' residues, respectively. We have also employed the slow binding kinetic approach to detail the mechanism of bovine trypsin and human factor Xa inhibition by the novel cytoplasmic antiproteinase. Inhibition of trypsin by the cytoplasmic antiproteinase was preceded by a two-step mechanism corresponding to the formation of an initial loose complex, followed by an isomerization step to a more stable, tight complex. The binding of the cytoplasmic antiproteinase to trypsin occurred with a second-order association rate constant of 2.8 x 10(6) M-1 s-1 and an overall equilibrium constant of 22.5 pM, demonstrating that the factor is a potent inhibitor of this proteinase. Under the appropriate conditions, the tight complex between trypsin and the cytoplasmic inhibitor was reversible, indicated by an exponential regeneration of proteinase amidolytic activity from the preformed complex. Therefore, the tight complex appears to be stabilized predominantly by reversible bonds that form between trypsin and the cytoplasmic inhibitor. In contrast to the inhibition of trypsin, the inhibition of factor Xa amidolytic activity by the cytoplasmic antiproteinase followed a single-step binding mechanism. The apparent first-order rate constant for factor Xa inhibition was found to increase as a linear function of the inhibitor concentration range studied. Formation of the inhibitory complex between factor Xa and the cytoplasmic antiproteinase occurred with a second-order association rate constant of approximately 1.3 x 10(5) M-1 s-1 and a equilibrium constant of 3.7 nM. These findings suggests that the cytoplasmic inhibitor may initially encounter significant energy barriers for proper alignment with the substrate binding cleft of factor Xa. However, once aligned, the reaction proceeds rapidly to a tight factor Xa.inhibitor complex that dissociates at a slow rate.
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PMID:Complementary DNA cloning and kinetic characterization of a novel intracellular serine proteinase inhibitor: mechanism of action with trypsin and factor Xa as model proteinases. 813 80

Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa). The ecotin gene was cloned by PCR, highly expressed in E. coli, and purified from the E. coli periplasm. The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM. The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1. Ecotin prolonged clotting time ca. 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively. Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin. Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration. Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca. 390 nM. FXa cleaved ecotin slowly at pH 4.0 between M84 and M85. Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively. The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.
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PMID:Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. 814 99


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