Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.5 (thrombin)
 33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Cathepsin B, a tissue (lysosomal) proteinase, and two humoral proteinases, plasmin and kallikrein, activate the latent collagenase ('procollagenase') which is released by mouse bone explants in culture. Other lysosomal proteinases (carboxypeptidase B, cathepsin C and D) and thrombin did not activate the procollagenase. Dialysis of the culture fluids against 3M-NaSCN at 4 degrees C and, for some culture fluids, prolonged preincubation at 25 degrees C also caused the activation of procollagenase. 2. In all these cases, activation of procollagenase involved at least two successive steps: the activation of an endogenous latent activator present in the culture fluids and the activation of procollagenase itself. 3. An assay method was developed for the endogenous activator. Human serum, bovine serum albumin, casein and cysteine inhibited the endogenous activator at concentrations that did not influence the collagenase activity. N-Ethylmaleimide and 4-hydroxy-mercuribenzoate stimulated the endogenous activator, but iodoacetate had no effect. 4. It is proposed that cathepsin B, kallikrein and plasmin may play a role in the physiological activation of latent collagenase and thus initiate degradation of collagen in vivo. This may occur whatever the molecular nature of procollagenase (zymogen or enzyme-inhibitor complex) might be.
 ...
PMID:Further studies on the activation of procollagenase, the latent precursor of bone collagenase. Effects of lysosomal cathepsin B, plasmin and kallikrein, and spontaneous activation. 19 17

1. A simple, highly sensitive, specific fluorometric method for the determination of chymotrypsin is described. 2. The new substrate utilized in this assay, N-glutaryl-glycyl-glycyl-l-phenylalanine beta-naphthylamide (GGPNA), is readily soluble in water, stable and highly specific for chymotrypsin. It is not degraded by a large excess of carboxypeptidase B, elastase, thrombin or plasmin and is virtually resistant to trypsin. 3. GGPNA is extremely sensitive to the action of chymotrypsin and permits detection of enzyme concentrations as low as 1 ng/ml. Linearity between enzyme concentration and fluorescence produced is maintained up to at least 3000 ng/ml. 4. alpha2-Macroglobulin-bound chymotrypsin hydrolyzes GGPNA at a rate about 2/3 of that exhibited by the free enzyme. 5. Bile pigments in amounts normally found in duodenal juice or traces of blood do not interfere with the assay. 6. GG PNA which releases beta-naphthylamine upon hydrolysis is suitable also for colorimetric and histological determination of chymotrypsin.
 ...
PMID:A new, highly sensitive and specific assay for chymotrypsin. 23 87

In the present study we have quantitatively characterized the interaction of purified human Glu- and Lys-plasminogen with intact and degraded fibrin by ligand-binding experiments using a radioisotopic dilution method and antibodies against human plasminogen. A fibrinogen monolayer was covalently linked to a solid support with polyglutaraldehyde and was treated with thrombin or with thrombin and then plasmin to respectively obtain intact and degraded fibrin surfaces. Under these conditions, a well-defined surface of fibrin is obtained (410 +/- 4 fmol/cm2) and, except for a 39-kDa fragment, most of the fibrin degradation products remain bound to the support. New binding sites for plasminogen were detected on the degraded surface of fibrin. These sites were identified as carboxy-terminal lysine residues both by inhibition of the binding by the lysine analogue 6-aminohexanoic acid and by carboxy-terminal end-group digestion with carboxypeptidase B. The binding curves exhibited a characteristic Langmuir adsorption isotherm saturation profile. The data were therefore analyzed accordingly, assuming a single-site binding model to simplify the analysis. Equilibrium dissociation constants (Kd) and the maximum number of binding sites (Bmax) were derived from linearized expression of the Langmuir isotherm equation. The Kd for the binding of Glu-plasminogen to intact fibrin was 0.99 +/- 0.17 microM and for degraded fibrin was 0.66 +/- 0.22 microM. The Kd for the binding of Lys-plasminogen to intact fibrin was 0.41 +/- 0.22 microM and for degraded fibrin was 0.51 +/- 0.12 microM.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Characterization of the binding of plasminogen to fibrin surfaces: the role of carboxy-terminal lysines. 167 72

A versatile, convenient assay for vertebrate collagenases has been developed using the fluorescent peptide substrate dansyl-Pro-Gln-Gly-Ile-Ala-Gly-D-Arg. This sequence resembles that of collagen at the site of cleavage but includes modifications designed to eliminate nonspecific hydrolysis by contaminating peptidases. Both human skin fibroblast and bovine corneal cell collagenases cleave the substrate specifically at the Gly-Ile bond. Plasmin, thrombin, trypsin, alpha-chymotrypsin, carboxypeptidase B, and bacterial collagenase do not cleave the substrate. Elastase and angiotensin converting enzyme display 20- and 400-fold less activity than the vertebrate collagenases, respectively, and cleave the peptide at different positions. The assay is performed by incubating a 5- to 25-microliters aliquot of trypsin-activated sample with an equal volume of 2 mM substrate overnight at 33 degrees C and pH 7.5. Thin-layer chromatography then separates the fluorescent product from the substrate in less than 20 min and allows the detection of subnanogram levels of collagenase. The assay is applicable to the screening of large numbers of samples under different conditions of pH and ionic strength and is readily adaptable for use in a variety of collagenase-dependent systems, such as assays for collagenase activating and/or inducing factors.
 ...
PMID:A convenient fluorescent assay for vertebrate collagenases. 301 20

In this study, the effect of sixteen different enzymes on serum C1 and its subcomponents was investigated. The sixteen enzymes could be divided into three groups. First, enzymes which activate native C1: trypsin (optimal concentration 2.4 x 10(-4) mM); alpha-chymotrypsin (2.3 x 10(3) mM); thrombin (1.0 x 10(-5) mM); plasmin (1.9 x 10(-5) mM); elastase (5.8 x 10(-5) mM); pronase (3.0 x 10(-6) mM). All these enzymes are serine esterase and activate native serum C1 bound to EAC4 at the given concentration within 10 min at 30 degrees C. Furthermore, native C1 inhibited by a pentosanpolysulfoester, Sp54, is unable to undergo the internal activation but can be externally activated by the serine esterases. Second, enzymes which do not activate native C1 but result in a dose and time-dependent loss of C1 activity: collagenase; pepsin; carboxypeptidase B. Third, enzymes which have no effect on C1 and C1: Lysozyme; neuraminidase; beta-galactosidase; L-amino acid oxidase; arginase; streptokinase, and acetylcholinesterase.
 ...
PMID:Activation of the first component of complement, C1: comparison of the effect of sixteen different enzymes on serum C1. 619 90

Prokallikrein was activated by trypsin and by alpha-chymotrypsin, but not by proteases, such as plasmin, thrombin, urokinase, carboxypeptidase B, papain, elastase, pepsin, and cathepsin D. Moreover, rat fresh serum did not activate prokallikrein. Maximum activation of prokallikrein by trypsin was obtained at the concentration of 10 micrograms to 1 mg per ml in PBS and that by alpha-chymotrypsin was at the concentration of 5 mg per ml. The enzymic properties of trypsin-activated and alpha-chymotrypsin-activated kallikreins were identical with those of active kallikrein in the kidney.
 ...
PMID:Activation of prokallikrein in the rat kidney by proteases. 637 43

1. Cleavage of the human antithrombin III--thrombin complex with [14C]methoxyamine hydrochloride results in inactive thrombin and 14C-labelled antithrombin III. 2. Discontinuous polyacrylamide-gel electrophoresis of the reduced dissociation fragments of the complex in the presence of sodium dodecyl sulphate reveals two antithrombin III bands that do not resolve during electrophoresis without reduction. The heavy band has the electrophoretic mobility of the native protein. The light band has an apparent mol.wt. that is approx. 4000 less than the molecular weight of native antithrombin III. 3. Treatment of the cleavage products of the complex with carboxypeptidase B yields 1 mumol of arginine, a new C-terminal amino acid, per mumol of thrombin dissociated. The results indicate that during formation of the antithrombin III--thrombin complex, the inhibitor is cleaved at an arginine--X bond; this arginine residue forms a carboxylic ester with the enzyme, while the excised polypeptide remains bound through a disulphide bridge(s).
 ...
PMID:The covalent nature of the human antithrombin III--thrombin bond. 721 42

Previous studies demonstrated that tissue plasminogen activator-induced fibrinolysis in vitro is retarded in the presence of prothrombin (II) activation and that the anticoagulant-activated protein C appears profibrinolytic by preventing the formation of thrombin (IIa)-like activity during fibrinolysis. To disclose the molecular connection between the generation of IIa and the inhibition of fibrinolysis, a lysis assay that is sensitive to the antifibrinolytic effect of II activation was developed and was used to purify a 60-kDa single-chain protein from human plasma. Because the lysis of a clot, produced from purified components, is retarded when this protein is present and when II activation occurs in situ, the protein was named TAFI (thrombin-activatable fibrinolysis inhibitor). TAFI is cleaved by IIa yielding 35-, 25-, and 14-kDa products. Amino-terminal sequence analyses identified TAFI as a precursor of a plasma carboxypeptidase B (CPB). Formation of the 35-kDa product correlates with both prolongation of lysis time and CPB-like activity. Prolongation of lysis time saturates at about 125 nM TAFI. Activated TAFI inhibits the activation of Glu-plasminogen but does not prolong the lysis of clots formed in the presence of Lys-plasminogen. 2-Guanidinoethylmercaptosuccinic acid, a competitive inhibitor of CPB, completely inhibits prolongation of lysis by activated TAFI in a purified system and the prolongation induced by II activation in barium-adsorbed plasma. This suggests that TAFI accounts for the antifibrinolytic effect that accompanies prothrombin activation and that activated protein C appears profibrinolytic by attenuating TAFI activation.
 ...
PMID:Purification and characterization of TAFI, a thrombin-activable fibrinolysis inhibitor. 778 9

Thrombin hydrolyzes the Arg156-Phe157 bond in pro-urokinase (pro-UK), two residues from the activation site, generating a two-chain form (thromb-UK) believed to have little activity and that is resistant to plasmin activation. The kinetic constants for thromb-UK against synthetic substrate (S2444) were found to be essentially identical to pro-UK. Against native plasminogen, thromb-UK had a lower Michaelis constant (KM) and a higher (2-fold) catalytic efficiency. However, this difference with pro-UK was nullified by carboxypeptidase B (CpB) treatment of thromb-UK to remove the C-terminal arginine on the A-chain. Plasminogen activation by thromb-UK was substantially promoted by fibrin fragment E-2 but not by other fibrin derivatives, a phenomenon previously observed with pro-UK. Similarly, clot lysis by thromb-UK was promoted by tissue plasminogen activator because their combined effect was synergistic. Fibrinogenolysis in plasma occurred at 80-fold the concentration of thromb-UK as pro-UK, reflecting the 90-fold greater plasmin resistance of thromb-UK. Addition of a CpB inhibitor to the plasma enhanced fibrinogenolysis by thromb-UK and pro-UK by approximately 16%, consistent with the promotion of both forms by certain C-terminal lysines. In conclusion, CpB-thromb-UK corresponds functionally to a plasmin resistant form of pro-UK, indicating that the catalytic site of the single-chain pro-UK is unaffected by thrombin cleavage. The effect of CpB indicates that the C-terminal Arg of thromb-UK slightly enhances its affinity for plasminogen. Thromb-UK has potential plasminogen-activating activity at surfaces where C-terminal lysines, functionally comparable to fragment E-2, are found.
 ...
PMID:The kinetics of plasminogen activation by thrombin-cleaved pro-urokinase and promotion of its activity by fibrin fragment E-2 and by tissue plasminogen activator. 842 4

TAFI (thrombin-activatable fibrinolysis inhibitor) is a recently discovered plasma protein that can be activated by thrombin-catalyzed proteolysis to a carboxypeptidase B-like enzyme that inhibits fibrinolysis. This work shows that the thrombin-thrombomodulin complex, rather than free thrombin, is the most likely physiologic activator. Thrombomodulin increases the catalytic efficiency of the reaction by a factor of 1250, an effect expressed almost exclusively through an increase in kcat. The kinetics of the reaction conform to a model whereby thrombin can interact with either TAFI (Km = 1.0 microM) or thrombomodulin (Kd = 8.6 nM), and either binary complex so formed can then interact with the third component to form the ternary thrombin-thrombomodulin-TAFI complex from which activated TAFI is produced with kcat = 1.2 s-1. This work also shows that activated TAFI down-regulates tPA-induced fibrinolysis half-maximally at a concentration of 1.0 nM in a system of purified components. This concentration of TAFI is about 2% of the level of the zymogen in plasma, which indicates that ample activated TAFI could be generated to very significantly modulate fibrinolysis in vivo. Therefore, TAFI in vitro and possibly in vivo defines an explicit molecular connection between the coagulation and fibrinolytic cascades, such that expression of activity in the former down-regulates the activity of the latter.
 ...
PMID:TAFI, or plasma procarboxypeptidase B, couples the coagulation and fibrinolytic cascades through the thrombin-thrombomodulin complex. 866 47


1 2 3 4 5 Next >>