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

---

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

cGMP-dependent protein kinase (cGMP kinase) has been implicated in the regulation of the cytosolic calcium level ([Ca2+]i). In Chinese hamster ovary (CHO) cells stably transfected with the cGMP kinase I alpha (CHO-cGK cells), cGMP kinase suppressed the thrombin-induced increase in inositol 1,4,5-trisphosphate and [Ca2+]i (Ruth, P., Wang, G.-X., Boekhoff, I., May, B., Pfeifer, A., Penner, R., Korth, M., Breer, H., and Hofmann, F. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 2623-2627). Cholecystokinin activated intracellular calcium release via a pertussis toxin (PTX)-insensitive pathway in CHO-cGK cells. cGMP kinase did not attenuate the CCK-stimulated [Ca2+]i. In contrast, cGMP kinase suppressed calcium influx stimulated by insulin-like growth factors 1 and 2 (IGF-1 and IGF-2) via PTX-sensitive pathways. The effects of PTX and cGMP kinase on [Ca2+]i were not additive. 8-Bromo-cGMP had no effect on [Ca2+]i stimulated by IGF-1 or IGF-2 in wild type CHO cells. These results suggested that cGMP kinase inhibited the different signaling pathways by the phosphorylation of a PTX-sensitive G protein. cGMP kinase phosphorylated the alpha subunits of Gi1, Gi2, and Gi3 in vitro. Phosphorylation stoichiometry was 0.4 mol of phosphate/mol of G alpha i1 after reconstitution of heterotrimeric Gi1 in phospholipid vesicles. The alpha subunit of Gi was also phosphorylated in vivo. These results show that cGMP kinase blocks transduction of distinct hormone pathways that signal via PTX-sensitive Gi proteins.
...
PMID:Cyclic GMP-dependent protein kinase blocks pertussis toxin-sensitive hormone receptor signaling pathways in Chinese hamster ovary cells. 772 18

Anhydrothrombin, a catalytically inactive derivative of thrombin in which dehydroalanine replaces the active-site serine, was prepared by a novel method. The active-site serine of thrombin was modified to dehydroalanine by promoting the beta-elimination of phenylmethylsulfonic acid from phenylmethylsulfonyl fluoride-inactivated thrombin under conditions in which the enzyme is unfolded. After the elimination reaction was quenched, the resulting anhydrothrombin was folded by diluting the denaturant, Gdn.HCl, to nondenaturing concentrations. Anhydrothrombin was purified by PAB affinity chromatography. Both native thrombin and anhydrothrombin were digested by cyanogen bromide, and the peptides from the region of the active-site serine (S205) were isolated by reverse-phase high-pressure liquid chromatography. Serine was present in the native thrombin peptide but absent from the anhydrothrombin peptide, as shown by amino acid analysis. This anhydrothrombin peptide was found to be 18.7 +/- 1.6 lower in mass units than the native peptide by electrospray mass spectrometry, in accord with the elimination of a water molecule. The anhydrothrombin preparation was monomeric, as determined by sedimentation equilibrium. Anhydrothrombin was used in a competitive titration of the complex of native thrombin with the leech saliva protein hirudin, a potent thrombin inhibitor, as measured by the recovery of thrombin amidolytic activity. This demonstrated that anhydrothrombin is capable of nativelike binding interactions with macromolecular ligands.
...
PMID:Preparation and characterization of anhydrothrombin. 775 77

The purpose of this study was to compare the ability of fresh and cryopreserved mononuclear cells to generate thrombin, induce fibrin formation and finally resolve the fibrin formed, when exposed to plasma. Peripheral blood mononuclear cells (PBM) from 4 donors were collected by gradient centrifugation on Lymfoprep, and cryopreserved in fetal calf serum and 10% dimethyl sulfoxide. Viability was tested by exclusion of trypan blue, as well as green/red fluorescence of fluorescein-diacetate and ethidium bromide (FDA/EB). Fresh and frozen-thawed cells were seeded, stimulated with lipopolysaccharide(LPS), and exposed to a standard heparinized overlay plasma. Plasma was harvested at intervals (0-7 days). Thrombin generation and fibrin formation were measured by quantification of prothrombin fragment (F1 + 2) and fibrinopeptide A (FPA) and the fibrinolytic capacity of the cells as the amount of fibrin (ogen) degradation products (FbDP and FgDP). Recovery of cells after thawing was about 80%, and the viability of fresh and cryopreserved PBM was > 95%. Compared to fresh, frozen cells fully retained their capability of Tissue Factor synthesis, leading to prothombinase activity (F1 + 2) and fibrin formation (FPA). In contrast, the fibrinolytic capacity of frozen-thawed cells were significantly reduced. As expected there were significant variations between the donors in all the parameters measured. We conclude that cryopreservation of human blood mononuclear cells is possible with maintainance of the potential of the cells to mediate coagulation in plasma upon LPS stimulation, whereas the fibrin resolving capacity apparently is reduced by the preservation procedure.
...
PMID:Procoagulant and profibrinolytic activities of cryopreserved human monocytes. 787 96

During the conversion of fibrinogen to fibrin, two amino-terminal fibrinopeptides A (FPAs) are cleaved by thrombin from each molecule. During early phases of conversion, fibrin intermediates lacking one of two FPAs (des A fibrin) are produced, the level of which depends on whether the FPA cleavage sequence from each molecule is random of concerted. Random cleavage of FPA would produce higher levels of des A fibrin at any thrombin concentration than would concerted cleavage, and the level of this intermediate product would have an important effect on the ultimate structure of the fibrin clot. Because evidence bearing on this subject is conflicting, we carried out experiments to assess the FPA release sequence from fibrinogen by thrombin or by an FPA-cleaving snake venom enzyme, Atroxin. At timed intervals the enzymatic reaction was terminated by precipitation with trichloroacetic acid, and the precipitate was then treated with cyanogen bromide to produce a dimeric amino-terminal fragment. These disulfide-linked amino-terminal fragments of fibrinogen, containing both, one, or neither FPA, were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their distribution quantified by densitometry. The rates of cleavage of the first FPA, k1, and of the second FPA, k2, were computed by fitting the data to equations for a consecutive chemical reaction. This analysis indicated that cleavage by either enzyme resulted in substantial amounts of des A fibrin intermediates. The ratio of the cleavage rates (k2/k1) was higher for thrombin (1.2 +/- 0.3) than it was for Atroxin (0.7 +/- 0.2) but indicates in both cases that the release rate of the second FPA is nearly the same as that of the first FPA.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Sequence of release of fibrinopeptide A from fibrinogen molecules by thrombin or Atroxin. 789 5

A fibrinogen-clotting enzyme (bothrombin) was purified from the venom of Bothrops jararaca. Bothrombin showed M(r) values of 33,000 under nonreducing and 35,000 under reducing conditions on SDS polyacrylamide gel electrophoresis and specific fibrinogen-clotting activity equivalent to 814-904 NIH alpha-thrombin units/mg. Diisopropyl fluorophosphate totally abolished its activity, but hirudin, a specific alpha-thrombin inhibitor, had negligible effect on bothrombin activity. Unlike alpha-thrombin, bothrombin split off fibrinopeptide A without releasing fibrinopeptide B. Bothrombin activated blood coagulation factor VIII, but its activity was about 950 times less than that of alpha-thrombin. Bothrombin did not induce aggregation or serotonin release of washed normal platelets by itself, but did aggregate platelets in the presence of exogenous fibrinogen. This latter activity was completely inhibited by either anti-glycoprotein (GP) IIb/IIIa monoclonal antibody (which blocks fibrinogen binding to GP IIb/IIIa) or anti-GP Ib monoclonal antibody (which specifically inhibits alpha-thrombin binding to GP Ib). Prostaglandin E1 (1 microM) and EDTA (10 mM) also abolished platelet aggregation without affecting clotting activity. Washed platelets from a patient with Bernard-Soulier syndrome did not respond to bothrombin even in the presence of exogenous fibrinogen, suggesting that the initial binding of bothrombin on platelets is GP Ib, but not a recently cloned thrombin receptor. The complete amino acid sequence of bothrombin was determined by analysis of (S)-pyridylethylated protein and peptides generated by digestion with cyanogen bromide and Achromobacter protease I, respectively. Bothrombin is composed of 232 amino acid residues and contains three Asn-linked oligosaccharide chains.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Purification and characterization of bothrombin, a fibrinogen-clotting serine protease from the venom of Bothrops jararaca. 811 Jul 87

The complete amino acid sequence of a thrombin-like enzyme with gyroxin activity isolated from the venom of the bushmaster snake Lachesis muta muta was determined by automated and DABITC/PITC microsequencing of the intact protein; fragments derived from it by separate cleavages with cyanogen bromide, iodosobenzoic acid and hydroxylamine; and peptides resulting from enzymatic digestions with trypsin, pepsin, chymotrypsin, and elastase. The protein, which is composed of 228 residues, contains four putative sites of N-linked glycosylation and exhibits significant sequence similarities with other serine proteases reported from snake venoms.
...
PMID:The complete amino acid sequence of a thrombin-like enzyme/gyroxin analogue from venom of the bushmaster snake (Lachesis muta muta). 835 84

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.
...
PMID:Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line. 840 7

Following stimulation of rabbit platelets with thrombin, phospholipase A2 (PLA2) activity increased in the Triton X-100-insoluble residue. Although the PLA2 activity was dependent on the protein content of the residue from the stimulated cells, the specific activity was higher than that in the case of unstimulated cells. The enzyme activity was inhibited by p-bromophenacyl bromide and increased significantly with 0.5-10 microM Ca2+. The enzyme hydrolyzed phospholipids having an arachidonoyl residue more effectively than ones with a linoleoyl residue. In addition, 70% of the enzyme activity was immunoprecipitated with a monoclonal antibody against cytosolic PLA2 of rabbit platelets, while it was inhibited by only 20% by an antibody that neutralizes the activity of group II PLA2. These results suggest an increase in the association of cytosolic PLA2 with cytoskeleton upon stimulation of rabbit platelets.
...
PMID:Evidence for an increase in the association of cytosolic phospholipase A2 with the cytoskeleton of stimulated rabbit platelets. 845 72

This work was carried out to decide whether a non-specific perturbation of the platelet membrane with exogenous amphiphiles affects protein phosphorylation in platelets, especially phosphorylation mediated by PKC. Effects of amphiphiles per se on protein phosphorylation were also recorded. (i) Sublytic concentrations of the differently charged model surfactants cetyltrimethylammonium bromide (CTAB), Zwittergent 3-16, sodium tetradecyl sulphate, and octaethyleneglycol hexadecyl ether, as well as chlorpromazine, and Triton X-100, did not affect the thrombin-induced, PKC-mediated phosphorylation of pleckstrin, whereas sphingosine blocked this phosphorylation. (ii) The sphingosine-mediated phosphorylation blockade is not related to a non-specific perturbation of the membrane, but can instead be attributed to specific properties of sphingosine. (iii) The amphiphiles, per se, had differential effects on protein phosphorylation at sublytic concentrations: a treatment with CTAB, Zwittergent 3-16, and sodium tetradecyl sulphate for 1 min led to phosphorylation of a 49-kDa protein, while treatment with sphingosine for 1 min led to a transient phosphorylation of the myosin light chain as well as a weak phosphorylation of pleckstrin.
...
PMID:Perturbation of the platelet plasma membrane is not sufficient for inhibition of thrombin-induced PKC-activity. 848 83

We report the identification, purification, and partial amino acid sequence of a novel serine proteinase inhibitor which is present in extracts from human placentas and in the cytosolic fraction of the leukemic cell line K562. Extracts from these tissues exhibited time-dependent inhibition of the serine proteinase thrombin. This activity was not accelerated by heparin and corresponded to a protein which formed a 67-kDa complex with 125I-thrombin. The complex was stable on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A cleaved and functionally inactive form of the protein was purified from placental tissue by chromatography on DEAE-Sepharose, followed by affinity chromatography on thrombin-Sepharose. Antibodies raised against the placental protein recognized the inhibitor from K562 cells and placental extract. Western blotting experiments using the antibody showed that the uncleaved inhibitor has a molecular mass of 38 kDa. Amino acid sequencing was performed on the purified protein. Sequences of peptides resulting from digestion with cyanogen bromide followed by Endoproteinase Lys-c confirmed that this is a novel inhibitor with significant homology to the serpin family.
...
PMID:Identification and purification of a novel serine proteinase inhibitor. 848 44


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---