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)

The tumor promoter phorbol myristate acetate (PMA) induces the production of the serine protease plasminogen activator (PA) in cultures of normal chick embryo fibroblasts (CEF) and synergistically enhances PA production in Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF). Following PMA treatment of serum-free RSVCEF cultures, PA induction is accompanied by distinct morphological changes, including enhanced cell clustering and the formation of dense cellular aggregates. These alterations in the morphology of the PMA-treated transformed cells are inhibited by several protease inhibitors, including leupeptin, NPGB, SBTI, benzamidine and DFP, the specific inhibitor of serine enzymes. A number of protease inhibitors are ineffective in preventing the PMA-induced morphological changes; these include inhibitors of trypsin, chymotrypsin, elastase, thrombin and, most importantly, plasmin. The use of a fluorescent substrate to assay PA directly demonstrated that the pattern of inhibiton of PA activity correlates exactly with the inhibition of morphological changes. The of 3H-DFP to label and characterize serine zymes in the culture fluid from PMA-treated cells further indicated that PA is the serine protease responsible for the morphological changes. Thus PA itself can catalytically alter cellular behavior in culture independent of plasminogen, until not its only known natural substrate.
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PMID:Phorbol ester-induced morphological changes in transformed chick fibroblasts: evidence for direct catalytic involvement of plasminogen activator. 22 74

The human lymphokine, leucocyte migration-inhibitory factor (LIF), appears to be a serine esterase and protease by virtue of its susceptibility to the irreversible enzyme inhibitor, phenylmethylsulfonyl fluoride (PMSF), and by the ability of arginine esters and amides to protect LIF against PMSF-induced inactivation. In this paper, three methods are described by which putative substrates for LIF may be investigated. Thus, molecules satisfying the substrate specificities of this lymphokine should (1) protect LIF against inactivation by PMSF, (2) reduce LIF activity in vitro on polymorphonuclear leucocytes, and (3) reduce the esterolytic activity of purified LIF-rich supernatants. The first two reactions were tested by means of the leucocyte migration agarose technique; the third reaction was tested by a sensitive enzyme assay using tritiated tosyl arginine methyl ester as substrate. Guanosine 3',5'-cyclic monophosphoric acid, which is capable of protecting LIF against PMSF-induced inhibition, also inhibited the esterolytic activity of the purified LIF preparation. Four synthetic oligopeptide substrates for trypsin, thombin and plasmin were investigated. Only one, the thrombin- and trypsin-specific benzoyl-phenylalanyl-valyl-agarine-p-nitroanilide, possessed high affinity for the LIF molecule and may therefore prove to be a potent substrate for this lymphokine.
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PMID:Substrate specificity of the human lymphokine leucocyte migration-inhibitory factor (LIF): radioenzymic assay and inhibition by cGMP. 22 50

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.
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PMID:A new, highly sensitive and specific assay for chymotrypsin. 23 87

A purified preparation of bovine thrombokinase (activated Factor X) loses the ability to hydrolyze TAME (p-toluenesulfonyl-L-arginine methyl ester) when it is incubated at 37 degrees in 0.25 M Tris. HCl buffer, pH 7.4 with lauroxypropyl biguanide, N1, N5-dimethyl, N1-lauroxypropyl biguanide, N1-p-chlorophenethyl, N5-phenethyl biguanide, or N1-methyl, N1-p-chlorobenzyl, N5-o,p-dichlorobenzyl biguanide. Activity is lost much more slowly when 0.15 M NaCl is also present. Lauroxypropyl biguanide is the most potent of the compounds tested, 0.22 mM causing thrombokinase to lose almost all of its activity in about 30 minutes at 37 degrees in pH 7.4 buffered saline. Topical bovine thrombin also loses activity when incubated with either of the lauroxypropyl biguanides but not with the diphenethyl or the dibenzyl compound. Instead, the latter biguanides accelerate thrombin's hydrolysis of TAME. The percent acceleration is not affected or only slightly decreased by the presence of 0.15 M NaCl or KCl, and it is also unaffected by incubating the enzyme with the compounds in buffered saline for 4 to 120 minutes. Purified bovine trypsin is stabilized by both lauroxypropyl and the diphenethyl biguanide when incubated at 37 degrees in pH 7.4 buffered saline for the 60 minute test period but neither compounds has any effect on its rate of hydrolysis of TAME. It is postulated that the enzymes first react rapidly and reversibly with all of the test biguanides and, depending upon the enzyme and the substrate, the rate of hydrolysis of the substrate is unaffected, accelerated or inhibited. The lauroxypropyl biguanides also undergo a second, slower reaction with both thrombokinase and thrombin that produces loss of enzymatic activity. The dibenzyl and diphenethyl biguanides also undergo this second slow reaction with thrombokinase but not with thrombin, and none of the biguanides undergo this second reaction with trypsin.
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PMID:The effects of biguanides on thrombokinase, thrombin and trypsin. 24 90

Proteolytic digestion of the human T lymphoblastoid cell line (Molt-4) and of peripheral blood lymphocytes by trypsin, chymotrypsin, and pronase results in a progressive, time-and dose-dependent diminution of T lymphocyte-sheep red bloock cell (SRBC) rosette formation, whereas thrombin, plasmin, collagenase, DNAse, and phospholipase have not effect. Complete abrogation of SRBC binding is achieved when lymphocytes (1 x 108/ml) are incubated with either trypsin or chymotrypsin at 10 mug/ml for 30 min, and greater than 50% abrogation is observed between 3 to 10 min. Preincubation of SRBC with the 10 min and 20 min lymphocyte digest supernatants inhibited their subsequent binding by normal T lymphocytes by as much as 64%. Thirty-minute digests were less inhibitory. Equivalent digests from several human B lumphoblastoid cell lines and from a non-rosetting clone of Molt-4 cells were not inhibitory. Polyacrylamide gel electrophoresis followed by elution of serial gel slices revealed four distinct inhibitory bands (I-IV) in the 20-min digest supernatant whereas only bands I-III and band IV were present in the 10-min and 30-min digest supernatants, respectively, suggesting progressive proteolysis of a distinct receptor. These experiments indicate that the binding of SRBC by human T lymphocytes represents a receptor-ligand interaction rather than a nonspecific electrical charge phe nomenon and that the receptor is a discrete molecular species which can be isolated from the surface of T but not B lymphocytes by limited enzymatic proteolysis.
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PMID:Recovery of soluble sheep erythrocyte receptor from the T lymphocyte surface by proteolytic cleavage. 30 Mar 98

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.
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PMID:[Specificity of the effect of thrombin on the chemoreceptors of the frog carotid labyrinth]. 30 Nov 3

The aggregation of platelets by the antibiotic, ristocetin, requires a plasma cofactor (VIII:vWF) and one or more specific binding sites on the platelet membrane. The interaction between VII:vWF and the platelet was examined using VIII:vWF labelled with 125I. In the presence of ristocetin (1.5 mg/ml), from 70 to 90% of the 125I-VIII:vWF became platelet-bound. By contrast, only 21% was bound with thrombin (2.5 microgram/ml), and 2.2% with buffer alone. Fractionation of the platelets revealed that peak radioactivity was present in the membrane fraction. Treatment of ristocetin-reacted platelets with either chymotrypsin, 100 microgram/ml, or trypsin, 75 microgram/ml, resulted in the partial release of the membrane-bound radioactivity. It is concluded that VIII:vWF binds to the platelet membrane in the presence of ristocetin.
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PMID:Platelet-binding of the von Willebrand factor. 30 91

Prostacyclin (PGI(2)) is an unstable prostaglandin which inhibits platelet aggregation and serotonin release and causes vasodilation. The PGI(2) activity produced by monolayers of cultured human endothelial cells and fibroblasts was measured by the ability of their supernates to inhibit platelet aggregation in platelet-rich plasma, or to inhibit thrombin-induced [(14)C]serotonin release from aspirin-treated, washed platelet suspensions. Monolayers of cultured human endothelial cells, stimulated with sodium arachidonate, thrombin, the ionophore A 23187, or trypsin, secreted PGI(2) into the supernatant medium. Monolayers of fibroblasts produced PGI(2) activity only when stimulated by arachidonate. "Resting," intact monolayers did not produce detectable PGI(2), nor did monolayers treated with ADP or epinephrine. Production of PGI(2) activity was abolished by treatment of the monolayers with indomethacin, tranylcypromine, or 15-hydroperoxy arachidonic acid. The PGI(2) activity of the supernates was destroyed by boiling or acidification. Inhibition of thrombin with diisopropylfluoro-phosphate, and of trypsin with soybean trypsin inhibitor, abolished the stimulation of PGI(2) production by these enzymes. Production of thrombin at a site of vascular injury could, by stimulating PGI(2) synthesis by endothelial cells adjacent to the injured area, limit the number of platelets involved in the primary hemostatic response and help to localize thrombus formation.
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PMID:Stimulation of endothelial cell prostacyclin production by thrombin, trypsin, and the ionophore A 23187. 36 56

A comparison of the primary structure of human thrombin with the structures of chymotrypsin, trypsin, elastase and factor Xabeta reveals several structural features which may be involved in the specificity of thrombin toward macromolecular substrates. Among the major structural differences noted in such a comparison are the insertions of five extended peptide regions in the primary structure of alpha-thrombin when compared to chymotrypsin. These insertions, which we refer to as "loops", have been designated A, B, C, D, and E. The A, B and C "loops" in human thrombin appear to be large enough to interact at or near the active active site if an alpha-thrombin-chymotrypsin three-dimensional structural homology is assumed. In beta-thrombin, the configuration of the A and B "loops" may be perturbed by proteolysis, and the ability of beta-thrombin to clot fibrinogen is thus reduced. Perturbation of the configuration of the C "loops" by proteolysis in the formation of gamma-thrombin may further reduce the ability of thrombin to bind fibrinogen.
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PMID:Thrombin: structural features related to specificity. 36 59

A 10-year-old boy had a severe lifelong hemorrhagic disorder that had necessitated more than 50 hospitalizations. Laboratory examination showed prolonged bleeding, clotting, partial thromboplastin, prothrombin, and thrombin times. These findings were due to a potent inhibitor of the thrombin-fibrinogen reaction. This inhibitor was similar to heparin in that it acted immediately and did not interfere with the coagulant activities of certain venoms. It differed from heparin in not being adsorbed to barium citrate or neutralized by protamine sulfate. The inhibitory effect was found in the alpha1-globulin fraction. It was identified immunologically and functionally as a double-banded alpha1-antitrypsin of a previously unreported phenotype. The inhibitory effects were depressed by trypsin and heterologous anti-alpha1-antitrypsin.
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PMID:Antithrombin Pittsburgh: an alpha1-antitrypsin variant causing hemorrhagic disease. 41 31


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