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)

Secretion of platelet granule constituents is closely associated with the phosphorylation of a cytosol polypeptide of Mr = 47,000 that we have called P47 (Haslam, R. J., Lynham, J. A., and Fox, J. E. B. (1979) Biochem. J. 178, 397-406). This polypeptide is a substrate of Ca2+-activated phospholipid-dependent protein kinase (Kawahara, Y., Takai, Y., Minakuchi, R., Sano, K., and Nishizuka, Y. (1980) Biochem. Biophys. Res. Commun. 97, 309-317). Two-dimensional gel electrophoresis of protein from human platelets that had been preincubated with 32Pi demonstrated the presence under control conditions of 2-3 major forms of P47 that contained very little 32P (pI values, 6.6-6.8) and, after induction of secretion with thrombin, their replacement by 7-9 highly labeled phosphorylated forms of P47 (pI values, 6.1-6.5). Native phosphorylated P47 was purified from thrombin-stimulated 32P-labeled platelets by ammonium sulfate fractionation and column chromatography on DEAE-cellulose, phenyl-Sepharose, and hydroxylapatite. The final 32P-labeled product was obtained in a yield of 20-25% and was purified about 400-fold relative to platelet lysate. This material was homogeneous on sodium dodecyl sulfate-polyacrylamide gel electrophoresis but, like the starting material, contained 7-9 separable phosphorylated components with different pI values. Purified phosphorylated P47 had a sedimentation coefficient (s20,w) of 3.57 S and a Stokes radius of 3.33 nm from which an Mr = 49,000 and a frictional ratio (f/f0) of 1.4 were calculated. These findings and failure to detect multimers after treatment of the protein with dimethyl suberimidate indicate that P47 normally exists as a monomer. The 32P-labeled phosphate present in purified P47 had the chemical stability of serine or threonine phosphoesters and analysis indicated the presence of 83% phosphoserine and 17% phosphothreonine. Limited proteolysis of purified 32P-labeled P47 by Staphylococcus aureus V8 protease generated a major unlabeled fragment (Mr = 23,500) and up to six labeled fragments (Mr = 24,700-14,800), the relative amounts of the latter depending on the extent of proteolysis. The same labeled fragments were obtained after proteolysis of each of the major phosphorylated components of P47, suggesting that these represent different phosphorylation states of variants of the same protein and that most or all of the phosphorylation sites are on a single 14,800-Da segment of the protein. The availability of pure native phosphorylated P47 should facilitate investigation of the physiological role of this protein in platelets.
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PMID:Purification and characterization of the 47,000-dalton protein phosphorylated during degranulation of human platelets. 688 23

Protein C is a precursor of plasma serine proteinases, and its active form inactivates specifically blood coagulation Factor V and Factor VIII. Since a specific and sensitive synthetic substrate for the activated protein C was not known, we studied its amidolytic activity toward 25 fluorogenic peptides of the type peptidyl-4-methylcoumaryl-7-amide (peptidyl MCA). The activated protein C, namely, bovine protein C activated by bovine alpha-thrombin, showed the highest activity toward Boc-Leu-Ser-Thr-Arg-MCA. The enzyme's Km and Kcat values for this substrate were calculated to be 3.3 x 10(-4) M and 8.4 s-1, respectively. Optimum conditions for measurement of activated protein C activity were studied with this substrate. Optimum pH was 8.5. For the maximum activity at pH 8.5, concentrations of 0.1 M NaCl and 1 mM CaCl2 had to be maintained in the reaction mixture. The fluorogenic peptide Boc-Leu-Ser-Thr-Arg-MCA was successfully applied to a simple and accurate assay of protein C during its purification.
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PMID:A new fluorogenic peptide substrate for vitamin K-dependent blood coagulation factor, bovine protein C. 689 48

Chicken antithrombin was purified from fresh chicken plasma by affinity chromatography using heparin-agarose, and its amino acid and carbohydrate compositions, amino-terminal sequence, inhibition of human thrombin, and immunological properties were studied and compared with previously studied mammalian antithrombins (human, pig, rabbit, and rat), and also with chick ovalbumin. Chicken antithrombin is a single-chain glycoprotein with a total carbohydrate content of 17.5%, including 6.0% N-acetylglucosamine, 8.7% hexose, and 2.8% N-acetylneuraminic acid. The molecular weight estimated from sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis was 60,000. The amino-terminal sequence has been determined as Ala-Pro-Tyr-Ala-Val-Glu-Asp-Ile-Cys-Thr-Ala-Lys-Pro-Thr-Asp-Ile-Pro-Val-Asn, which is highly homologous to the terminal sequences of mammalian antithrombins, although the first 4 residues are quite different from those of mammalian species. Chicken antithrombin showed a stoichiometric inhibition against thrombin. The apparent dissociation constant (K1) for the complex was 6.4 X 10(-8) M. No immunological cross-reactivity was observed between chicken and mammalian antithrombins. Ovalbumin, which Hunt and Dayhoff (Biochem. Biophys. Res. Commun. 95, 864-871, 1980) proposed should be grouped in the same superfamily as antithrombin, showed neither immunological cross-reactivity with antithrombin or with its carboxymethylated derivative, nor any effect on the thrombin-antithrombin interaction. Ovalbumin showed no inhibitory effect on porcine elastase, either.
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PMID:Chicken antithrombin. Isolation, characterization, and comparison with mammalian antithrombins and chicken ovalbumin. 692 Mar 85

The amino-terminal 173 residues of the murine histocompatibility antigen H-2Kb have been assigned by using radiochemical methodology. The complete sequence of an 86 residue glycopeptide (CN-Ib), which is one of the five major CNBr fragments of Kb, was determined by analysis of peptides obtained from digests using thrombin and V8 staphylococcal protease. Complete sequences were obtained for the three large thrombic peptides, and these were aligned by using peptides from the V8 protease digest. Alignment of the CNBr fragments was carried out by using [35S]Met-labeled peptides from a tryptic digest of the papain-cleaved H-2Kb molecule. Positive identification was possible for all the common amino acids except Asp (Asp) which was indirectly assigned and which is designated in italics. The sequence obtained in our studies was Gly-Pro-His-Ser-Leu-Arg-Tyr-Phe-Val-Thr-Ala-Val-Ser-Arg-Pro-Gly-Leu-Gly-Glu-Pro-Arg-Tyr-Met-Glu-Val-Gly-Tyr-Val-Asp-Asp-Thr-Glu-Phe-Val-Arg-Phe-Asp-Ser-Asp-Ala-Glu-Asn-Pro-Arg-Tyr-Glu-Pro-Arg-Ala-Arg-Trp-Met-Glu-Gln-Glu-Gly-Pro-Glu-Tyr-Trp-Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly-Asn-Glu-Gln-Ser-Phe-Arg-Val-Asp-Leu-Arg-Thr-Leu-Leu-Gly-Tyr-Tyr-(Asn)-Gln-Ser-Lys-Gly-Gly-Ser-His-Thr-Ile-Gln-Val-Ile-Ser-Gly-Cys-Glu-Val-Gly-Ser-Asp-Gly-Arg-Leu-Leu-Arg-Gly-Tyr-Gln-Gln-Tyr-Ala-Tyr-Asp-Gly-Cys-Asp-Tyr-Ile-Ala-Leu-Asn-Glu-Asp-Leu-Lys-Thr-Trp-Thr-Ala-Ala-Asp-Met-Ala-Ala-Leu-Ile-Thr-Lys-His-Lys-Trp-Glu-Gln-Ala-Gly-Glu-Ala-Glu-Arg-Leu-Arg-Ala-Tyr-Leu-Glu-Gly-Thr-Cys-Val-Glu-Trp-Leu-Arg-Arg-Tyr-Leu-Lys. These data represent the longest reported amino acid sequence determined by utilizing radiochemical methodology and provide the first extensive information on the primary structure of murine histocompatibility antigens.
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PMID:Primary structure of murine major histocompatibility complex alloantigens: amino acid sequence of the amino-terminal one hundred and seventy-three residues of the H-2Kb glycoprotein. 698 68

A clotting enzyme associated with the hemolymph coagulation system of Japanese horseshoe crab (Tachypleus tridentatus) was highly purified from the amebocyte lysate. The method for purification consisted of gel-filtration of the lysate on a pyrogen-free Sepharose CL-6B column and affinity chromatography of the endotoxin-treated clotting enzyme on a benzamidine-Sepharose 4B column. Through these procedures, about 3 mg of the purified enzyme was obtained from 70 ml of the lysate and about 390-fold purification was achieved. The purified preparation was found to give a single major band, respectively, on polyacrylamide-gel disc electrophoresis at pH 3.2 in the presence of 6 M urea and on sodium dodecyl sulfate (SDS)-gel electrophoresis in the presence and absence of 2-mercaptoethanol. It also gave a single symmetrical peak on QAE-Sephadex A-25 column chromatography. The molecular weight of the clotting enzyme was estimated to be approximately 42,000 for the unreduced sample by SDS-gel electrophoresis. For the reduced sample, it was 30,000, suggesting that the protein consists of plural polypeptide chains bridged by disulfide(s). The Tachypleus clotting enzyme was a glycoprotein, as shown by the positive periodic acid-Schiff reaction for the protein band on SDS-gel and the amino acid analysis. The purified clotting enzyme transformed Tachypleus coagulogen to coagulin gel and hydrolyzed a chromogenic peptide substrate, Tos-Ile-Glu-Gly-Arg-p-nitroanilide for Factor Xa, liberating p-nitroaniline. The enzyme was sensitive to DFP and benzamidine. It was also inhibited partially by PCMB. Antithrombin III and alpha 2-plasmin inhibitor (alpha 2-antiplasmin) were very effective inhibitors of this enzyme among ten kinds of naturally occurring proteinase inhibitors tested. The clotting enzyme had a restricted specificity towards protein substrates and activated only prothrombin among plasma zymogens including Factor IX, Factor X, fibrinogen, plasminogen and prekallikrein. The cleavage sites of bovine prothrombin for this enzyme were the same Arg-Thr and Arg-Ile linkages as those for Factor Xa, resulting in the formation of alpha-thrombin. These results indicate that the horseshoe crab clotting enzyme is a Factor Xa-like serine proteinase rather than alpha-thrombin. It seems likely that the Tachypleus clotting enzyme is a prototype of mammalian serine proteinases participating in blood coagulation.
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PMID:A clotting enzyme associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus): its purification and characterization. 714 17

The NH2-terminal 98 amino acid residues of the murine histocompatibility antigen H-2Db have been assigned using radiochemical methodology. This represents the first extensive, continuous sequence information for a histocompatibility antigen encoded by the H-2D locus and allows comparison with the recently determined amino acid sequence of the H-2Kb molecule. The amino acid sequence was obtained from the sequences of three CNBr peptides, CN-E, CN-D, and CN-B, which comprise residues 1-5, 6-52, and 53-98, respectively. The amino acid sequence of CN-E was determined directly while the sequences of CN-D and CN-B were determined by NH2-terminal sequence analyses and sequence determinations of peptides produced by thrombin, staphylococcal V8 protease, and trypsin cleavage. Alignment of the CNBr peptides was accomplished by NH2-terminal sequence analysis of the H-2Db papain fragment (CN-E to CN-D) and by analyzing peptides from a tryptic digest of the intact H-2Db molecule. Positive identification was possible for all amino acids except Asp and Asn-86 which were indirectly assigned (in italics). The sequence obtained was Gly-Pro-His-Ser-Met-Arg-Tyr-Phe-Glu-Thr-Ala-Val-Ser-Arg-Pro-Gly-Leu-Glu-Glu-Pro -Arg-Tyr-Ile-Ser-Val-Gly-Tyr-Val-Asp-Asn-Lys-Glu-Phe-Val-Arg-Phe-Asp-Ser-Asp-Ala-Glu-Asn-Pro-Arg-Tyr-Glu-Pro-Arg-Ala-Pro-Trp-Met-Glu-Gln-Glu-Gly-Pro-Glu-Tyr-T rp-Glu-Arg-Glu-Thr-Gln-Lys-Ala-Lys-Gly-Gln-Glu-Gln-Trp-Phe-Arg-Val-Ser-Leu-Arg-Asn-Leu-Leu-Gly-Tyr-Tyr-Asn-Gln-Ser-Ala-Gly-Gly-Ser-His-Thr-Leu-Gln-Gln-Met.
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PMID:Primary structure of murine major histocompatibility complex alloantigens. Amino acid sequence of the NH2-terminal ninety-eight residues of the H-2Db glycoprotein. 722 4

The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role for tyrosine and serine/threonine kinases has been proposed. Since mitogen-activated protein (MAP) kinase is activated by insulin through phosphorylation on both tyrosine and threonine residues, we investigated whether MAP kinase and its upstream regulator, p21ras, are involved in insulin-mediated glucose transport. We did this by examining the time- and dose-dependent stimulation of glucose uptake in relation to the activation of Ras-GTP formation and MAP kinase by thrombin, epidermal growth factor (EGF), and insulin in 3T3-L1 adipocytes. Ras-GTP formation was stimulated transiently by all three agonists, with a peak at 5 to 10 min. Thrombin induced a second peak at approximately 30 min. The activation of p21ras was paralleled by both the phosphorylation and the activation of MAP kinase: transient for insulin and EGF and biphasic for thrombin. However, despite the strong activation of Ras-GTP formation and MAP kinase by EGF and thrombin, glucose uptake was not stimulated by these agonists, in contrast to the eightfold stimulation of 2-deoxy-D-[14C]glucose uptake by insulin. In addition, insulin-mediated glucose transport was not potentiated by thrombin or EGF. Although these results cannot exclude the possibility that p21ras and/or MAP kinase is needed in conjunction with other signaling molecules that are activated by insulin and not by thrombin or EGF, they show that the Ras/MAP kinase signaling pathway alone is not sufficient to induce insulin-mediated glucose transport.
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PMID:Activation of the Ras/mitogen-activated protein kinase signaling pathway alone is not sufficient to induce glucose uptake in 3T3-L1 adipocytes. 751 Dec 5

A murine monoclonal antibody (MAb) raised against a covalent antithrombin-heparin complex was used to probe the conformational change resulting when the serpin antithrombin binds to heparin. This MAb completely inhibited the progressive activity of antithrombin against thrombin. However, although the MAb remained bound to antithrombin in the presence of heparin, it did not significantly inhibit heparin cofactor activity against thrombin, and increasing concentrations of the antithrombin-binding pentasaccharide progressively unblocked the inhibitory action of the MAb. The MAb bound to antithrombin without affecting either heparin-binding affinity or heparin-induced fluorescence enhancement, and it did not convert antithrombin from inhibitor to substrate. The MAb failed to interact with reduced and S-carboxymethylated antithrombin, indicating the conformational nature of its epitope. Antithrombin variants with N-terminal substitutions (Arg47-->Cys or His, Leu99-->Phe, Arg129-->Gln) modifying heparin binding, and C-terminal substitutions affecting the reactive site (Arg393-->Cys) or resulting in substrate-variant antithrombin (Ala384-->Pro), were all recognized normally, as were normal reactive site cleaved antithrombin and the thrombin-antithrombin complex. However, interaction of the MAb with antithrombin was reduced by several substitution mutations (Phe402-->Cys, Phe402-->Ser, Phe402-->Leu, Ala404-->Thr, Pro407-->Thr) in the 402-407 sequence which codes for amino acid residues of strand 1C and the polypeptide leading to strand 4B. Pro429-->Leu also blocks recognition [Olds et al. (1992) Blood 79, 1206-1212], and this residue is believed to be spatially approximated to strand 1C.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Conformational change in antithrombin induced by heparin probed with a monoclonal antibody against the 1C/4B region. 751 82

Thrombin and the thrombin receptor agonist peptide (TRAP) caused a rise in intracellular calcium concentration ([Ca2+]i) in the human osteoblast-like cell line Saos-2. Striking differences in the [Ca2+]i signals elicited by these agonists were revealed. In cell populations, thrombin induced a transient increase in [Ca2+]i while TRAP caused a biphasic [Ca2+]i response consisting of an initial peak and a sustained plateau phase. In individual cells, thrombin mainly caused a single [Ca2+]i transient while TRAP induced repetitive [Ca2+]i spikes. Neither tyrosine phosphorylation, cAMP-dependent phosphorylation, nor pertussis toxin-sensitive G proteins appeared to be involved in thrombin receptor [Ca2+]i signaling in this cell line. However, the sustained [Ca2+]i response caused by TRAP was converted into a transient, thrombin-like response by pretreatment with serine/threonine phosphatase inhibitors. Pretreatment with the phorbol ester phorbol 12-myristate 13-acetate (PMA) abrogated thrombin receptor [Ca2+]i signaling, and TRAP-induced Ca2+ entry was inhibited by the acute treatment with PMA. In contrast, Ca2+ entry stimulated by thapsigargin was not sensitive to agents affecting serine/threonine phosphorylation. The observation that thrombin and TRAP, despite being agonists for a common receptor, induce dissimilar [Ca2+]i responses indicates that binding of TRAP alone is insufficient to fully regulate the thrombin receptor in Saos-2 cells.
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PMID:Differences in intracellular calcium signaling after activation of the thrombin receptor by thrombin and agonist peptide in osteoblast-like cells. 751 33

Activated platelets expressing P-selectin on their surface are known to adhere to monocytes and neutrophils. We examined the possibility that the leukocytes were activated by their adhesion to activated platelets and demonstrated that P-selectin-dependent platelet adhesion to neutrophils and monocytes induced production of extracellular superoxide anion (O2-) by these leukocytes. Leukocyte membrane glycoproteins containing Ser/Thr-linked carbohydrate chains were responsible for the signal reception leading to the leukocyte activation. Cytokines were shown to influence these processes. For example, treatments of neutrophils with interleukin-8 (IL-8) or granulocyte colony-stimulating factor (G-CSF) potentiated the P-selectin-induced O2- production. Furthermore, interleukin-1 (IL-1) and interferon-gamma (IFN-gamma) induced surface expression of P-selectin on platelets in the presence of a low concentration of thrombin and consequently enhanced their adhesion capacity to leukocytes. These results indicated that the adhesion of activated platelets to the leukocytes through the interaction between P-selectin and its carbohydrate ligand, sialyl Lewis X (LeX), was a crucial step for the activation of leukocyte function and supported the notion that activated platelets were actively involved in the inflammatory processes.
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PMID:Induction of superoxide anion production from monocytes an neutrophils by activated platelets through the P-selectin-sialyl Lewis X interaction. 752 17


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