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:3.4.21.5 (thrombin)
33,306 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two proteinases (2A and 2B) purified from the granular fraction of horse blood leucocytes degrade casein (Km values 12.8 and 6mg/ml respectively) with maximum activity at pH 7.4 and in the presence of 2m-urea. Urea-denatured haemoglobin, fibrinogen, albumin and resorcin/fuchsin-stained elastin are digested at a slower rate. The enzymes hydrolyse synthetic substrates of elastase, N-benzyloxycarbonyl-L-alanine 4-nitrophenyl ester (Km 0.114 and 0.178 mM) and N-acetyl-tri-L-alanine methyl ester (Km 5.55 and 0.98 mM), but they do not hydrolyse synthetic substrates of trypsin, chymotrypsin and thrombin. The examined proteinases are completely inhibited by 2 mM-di-isopropyl phosphorfluoridate and show a sensitivity to butyl and octyl isocyanates similar to that of pancreatic elastase. The pH-dependence of their photoinactivation in the presence of Rose Bengal indicates the presence of histidine in the active centre. Proteinase 2A rather insensitive to iodination by IC1 as is pancreatic elastase, whereas proteinase 2B is totally inactivated after incorporation of five iodine atoms per enzyme molecule.
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PMID:Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes. 0 9

The effect of thrombin-like snake venom proteases (Ancrod of Agkistrodon rhodostoma and Batroxobins of Bothrops moojeni and Bothrops marajoensis) on skeletal muscle actin was studied and compared to the thrombic cleavage of this protein. Only EDTA-pretreated G- and F-actin were split by thrombin and Ancrod, while Batroxobins hydrolyzed native G-actin, too. The time course of digestion was followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. A split product of 37 500 daltons appeared first which was cleaved further resulting in three lower molecular weight fragments. The sodium dodecyl sulfate gel pattern of thrombic fragmentation was well distinguishable from those caused by Ancrod and Batroxobins. The first split products of Batroxobin digestion--a smaller peptide and the 37 500 dalton fragment--were isolated and by estimating their N-, and C-terminal end groups and amino acid compositions the peptide bond hydrolyzed first was located in the primary structure of actin. It was established that while thrombin split off two actino-peptides (at Arg(28)-Ala(29) and Arg(39)-His(40) from the N-terminal end of the molecule only Arg(39)-His(40) was cleaved by Batroxobins.
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PMID:Fragmentation of actin by thrombin-like snake venom proteases. 42 15

Protein C is a vitamin K-dependent protein, which exists in bovine plasma as a precursor of a serine protease. In this study, protein C was isolated to homogeneity from human plasma by barium citrate adsorption and elution, ammonium sulfate fractionation, DEAE-Sephadex chromatography, dextran sulfate agarose chromatography, and preparative polyacrylamide gel electrophoresis. Human protein C (M(r) = 62,000) contains 23% carbohydrate and is composed of a light chain (M(r) = 21,000) and a heavy chain (M(r) = 41,000) held together by a disulfide bond(s). The light chain has an amino-terminal sequence of Ala-Asn-Ser-Phe-Leu- and the heavy chain has an aminoterminal sequence of Asp-Pro-Glu-Asp-Gln. The residues that are identical to bovine protein C are underlined. Incubation of human protein C with human alpha-thrombin at an enzyme to substrate weight ratio of 1:50 resulted in the formation of activated protein C, an enzyme with serine amidase activity. In the activation reaction, the apparent molecular weight of the heavy chain decreased from 41,000 to 40,000 as determined by gel electrophoresis in the presence of sodium dodecyl sulfate. No apparent change in the molecular weight of the light chain was observed in the activation process. The heavy chain of human activated protein C also contains the active-site serine residue as evidenced by its ability to react with radiolabeled diisopropyl fluorophosphate. Human activated protein C markedly prolongs the kaolin-cephalin clotting time of human plasma, but not that of bovine plasma. The amidolytic and anticoagulant activities of human activated protein C were completely obviated by prior incubation of the enzyme with diisopropyl fluorophosphate. These results indicate that human protein C, like its bovine counterpart, exists in plasma as a zymogen and is converted to a serine protease by limited proteolysis with attendant anticoagulant activity.
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PMID:Human plasma protein C: isolation, characterization, and mechanism of activation by alpha-thrombin. 46 91

Plasma fibrinopeptide B (Bbeta1-14 or FPB) immunoreactivity was studied by radioimmunoassay in patients who received intrauterine infusion of hypertonic saline to terminate pregnancy. FPB immunoreactivity increased with thrombin treatment (TIFPB) suggesting the presence of a larger FPB-containing peptide, since purified FPB is not altered by thrombin, whereas thrombin increases the immunoreactivity of Bbeta1-42 (which includes FPB) 10-fold. TIFPB immunoreactivity in plasma, drawn 4 h after hypertonic saline infusion eluted from Sephadex G-50 similarly to isolated Bbeta1-42. Streptokinase, incubated with normal plasma progressively generated TIFPB immunoreactivity, which showed a major component which eluted from Sephadex G-50 similarly to Bbeta1-42. Streptokinase generated TIFPB much more rapidly in reptilase-treated plasma that contains fibrin I, (which still includes FPB), indicating that fibrin I is preferred over fibrinogen as a substrate for plasmin cleavage of arginine (Bbeta42)-alanine (Bbeta43). Serial studies were then made in 10 patients receiving intrauterine hypertonic saline. Fibrinopeptide A (FPA) levels rose immediately, reached a peak between 1 and 2 h, were declining at 4 h, and were normal at 24 and 48 h. TIFPB levels rose slightly in the 1st h, reached a peak at 4 h, and had returned to base-line values at 24 h. Serum fibrinogen degradation product levels were unchanged at 1 h, reached their highest level at 4 h, and were still markedly elevated at 24 and 48 h. Fibrinogen levels dropped slightly being lowest at 4 and 24 h. Platelet counts declined in parallel with the fibrinogen levels over the first 4 h, but continued to decrease through 48 h. Beta thromboglobulin (betaTG) levels generally paralleled FPA levels whereas platelet factor 4 (PF4) levels showed only slight changes. The data indicate that immediately after intrauterine hypertonic saline infusion thrombin is formed that cleaves FPA from fibrinogen to produce fibrin I and releases betaTG and PF4 from platelets. Later plasmin cleaves Bbeta1-42 from fibrin I to produce fragment X, which is further degraded to form serum fibrinogen degradation products. This sequence of proteolysis indicates that plasmin action on fibrin I serves as a mechanism that regulates fibrin II formation by removing the Bbeta chain cleavage site, which is required for thrombin action in converting fibrin I to fibrin II.
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PMID:Sequence of fibrinogen proteolysis and platelet release after intrauterine infusion of hypertonic saline. 50 Aug 18

Synthetic procedures have been developed for the preparation of peptides of arginine chloromethyl ketone and applied in the preparation of affinity labels which correspond to the -Pro-Phe-Arg- C terminus of bradykinin, a physiological cleavage site of kallikrein in kininogen. Two such reagents, Ala-Phe-ArgCH2C1 and Pro-Phe-ArgCH2C1, proved to be highly effective as well as selective affinity labels for human plasma kallikrein. For example, Pro-Phe-ArgCH2C1 inactivates plasma kallikrein 50% in 24 min at a concentration of 2 x 10(-8)M, while other trypsin-like proteases are less susceptible in inactivation than kallikrein, differing by a factor of 48 for plasmin and factors of 10(2)-10(5) for factor Xa, thrombin, and urokinase. The affinity of human plasma kallikrein for Ala-Phe-ArgCH2C1 (Ki = 0.078 micron) is about 60 times that for Ala-Phe-LysCH2C1(Ki = 4.9 micron), whereas human plasmin exhibits about the same affinity for the former affinity label (Ki = 1.3 micron) as for the latter (Ki = 0.83 micron). The rate constants for the irreversible step of the affinity labeling reaction, k2, are similar for affinity labels tested with the individual proteases: 0.35 min-1 for plasma kallikrein and 0.18 min-1 for plasmin.
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PMID:Synthesis of peptides of arginine chloromethyl ketone. Selective inactivation of human plasma kallikrein. 72 86

Factor XII was purified approximately 14 000-fold from bovine plasma by ammonium sulfate fractionation followed by heparin-agarose, DEAE-Sephadex, CM-cellulose, arginine-agarose, and benzamidine-agarose column chromatography. By this method, about 15 mg of protein was purified from 15 L of plasma with an overall yield of 18%. The purified protein was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino-terminal analysis. Bovine factor XII is a glycoprotein with a mol wt of 74 000 as determined by sedimentation equilibrium centrifugation. It contains 13.5% carbohydrate including 3.4% hexose, 4.7% N-acetylhexosamine, and 5.4% N-acetylneuraminic acid. Factor XII is a single polypeptide chain with an NH2-terminal sequence of Thr-Pro-Pro-Trp-Lys-Gly-Pro-?-Lys-His. This sequence is homologous to the reactive-site regions of a number of protease inhibitors. The amino acid sequence of a carboxyl-terminal fragments prepared by cyanogen bromide digestion was found to be Leu-Cys-Ala-Gly-Phe-Leu-Glu-Gly-Gly-Thr-Asp-Ala-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro-Leu-Val-Cys-Glu-Asp-Glu. This sequence is homologous with the active site of a number of plasma serine proteases including thrombin, factor IXa, factor Xa, and plasmin. These data indicate that bovine factor XII is a precursor to a serine enzyme with an inhibitor sequence and a catalytic site located in the same single polypeptide chain.
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PMID:Isolation and characterization of bovine factor XII (Hageman factor). 86 Dec 10

Coagulation Factor VII from bovine plasma is a glycoprotein containing a single peptide chain. The NH2-terminal sequence of Ala-Asx-Gly-Phe-Leu- is homologous with the NH2 termini of prothrombin, Factor IX, and the light chain of Factor X. Factor Xa in the presence of calcium ions and phospholipid cleaves Factor VII at an Arg-Ile bond in the sequence Arg-Ile-Val-Gly-Gly-, producing a two-chain molecule with at least 85 times the coagulant activity of single-chain Factor VII and a new NH2-terminal sequence homologous with the corresponding chains of thrombin, Factor IXa and Factor Xa. A second slower cleavage at an Arg-Gly bond destroys Factor VII activity. Bovine Factor VII, unlike prothrombin, Factor IX, and Factor X, is rapidly inhibited by diisopropylphosphorofluoridate (iPr2PF). [3H]iPr2PF is readily incorporated into one-chain, two-chain, and three-chain forms of Factor VII up to ratios of approximately 0.9 moles of [3H]diisopropylphosphate per mole of protein. The radioactive peptides generated from each form of [32P]iPr2PF-inhibited Factor VII by tryptic and thermolytic digestion were found to migrate together on paper electrophoresis. This indicates that the iPr2PF is incorporated stoichiometrically into the same specific site in each form.
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PMID:Mechanism of activation of bovine factor VII. Products of cleavage by factor Xa. 95 65

A procedure for the preparation of highly purified sheep prothrombin is described. The purified zymogen, when subjected to disc gel electrophoresis in polyacrylamide, gave rise to one single band. Only alanine was found as N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. The isoelectric point, as determined by isoelectric focusing in polyacrylamide gel slab, was shown to be 4.9-5.0. Non-chromatographed, but not the purified zymogen, could be converted into active thrombin in half-saturated trisodium citrate seeded with thrombin. Pure sheep prothrombin showed 5.6% of neutral sugars and the following amino acid composition: Ala35, Arg44, Asx54-55, -Cys24, Glx72, Gly53-54, His8, Ile19, Leu45, Lys31, Met7, Phe23, Pro36, Ser34, Thr29-29, Trp16, Tyr19 and Val33, which accounts for a molecular weight of about 66 000 (amino acids only). The molecular weight as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis after reduction by 2-mercaptoethanol, was shown to be 77 000 +/- 3000 (carbohydrates included).
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PMID:Sheep prothrombin: purification and partial characterization. 99 97

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.
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PMID:The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit. 115 Jun 58

Digestion of calf thymus H1 histone with thrombin cleaves the molecule at the sequence -(Pro)-Lys-Lys-Ala-, corresponding to a point approximately 122 residues from the N-terminus (about 56% along the molecule). The N-terminal fragment is shown by proton nuclear magnetic resonance (NMR) to possess the globular structure of the intact histome H1 molecule, whereas the C-terminal fragment appears to possess little or no structure. The N-terminal fragment separates into two peaks on an ion-exchange column, one of which is shown to originate from a single subfraction of calf thymus histone H1 and the other to originate from the other subfractions, by detailed comparison of the NMR spectra. It thus seems that the structure of the H1 histone in solution under physiological conditions consists of a globular head with a highly basic random coil tail. It is suggested that the globular head has a specific binding site on the subunit structure of the chromosome.
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PMID:Studies on the role and mode of operation of the very-lysine-rich histone H1 in eukaryote chromatin. The isolation of the globular and non-globular regions of the histone H1 molecule. 124 89


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