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)

Anthranilate synthase is involved in tryptophan (Trp) biosynthesis. Functional expression of subunit I from Arabidopsis (ASA1) was achieved in bacteria as a protein fused with glutathione S-transferase (GST). The active product was purified in a single step on a glutathione-Sepharose column. The Vmax (45 nmol min-1mg-1), the apparent K(M) for chorismate (180 microM), and the feedback inhibition by Trp (complete inhibition by 10 microM Trp) of the purified fusion product (GST-ASA1) were comparable to anthranilate synthase purified from plants. Polyclonal antibodies raised against the fusion project and purified by affinity chromatography on a GST-ASA1-Sepharose column cross-reacted with a 61.5-kD protein in a partially purified anthranilate synthase preparation from corn seedlings. GST-ASA1 cleavage by thrombin, as well as site-directed mutagenesis modifications of the Trp allosteric site, inactivated the recombinant protein.
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PMID:Functional expression of Arabidopsis thaliana anthranilate synthase subunit I in Escherichia coli. 797 19

3',3",5',5"-Tetraiodophenolsulfonephthalein (I4PSP) stimulated both Ca2+ transient levels and aggregation in response to thrombin in platelets. Ca2+ uptake into skeletal sarcoplasmic reticulum (SR) was inhibited by I4PSP (IC50, 1.8 microM) whereas that of red blood cell ghosts was not affected by it. Furthermore, I4PSP inhibited the SR Ca(2+)-ATPase activity (IC50, 1.1 microM). Kinetic analysis of the inhibitory effects of I4PSP reveals that I4PSP shows an inhibition of noncompetitive and competitive type with respect to low and high concentrations of ATP, respectively. The mode of inhibition by I4PSP is an uncompetitive type with respect to Ca2+. I4PSP decreased the decomposition rate of the phosphorylated enzyme intermediate (EP). The change in the tryptophan fluorescence of SR Ca(2+)-ATPase induced by ATP was reduced by I4PSP indicating inhibition of the EP transition from Ca2E1P to E2P. The concentration-inhibitory response curves for I4PSP in Ca(2+)-ATPase activities and fluorescence changes were closely correlated. These results suggest that I4PSP slows down the structure transition from Ca2E1P to E2P, resulting in inhibition of the Ca(2+)-ATPase activity. On the basis of these results, it is suggested that the stimulation of Ca2+ transient levels in platelets and their aggregation in response to thrombin are due to the inhibition of Ca2+ pump in intracellular Ca2+ stores by I4PSP.
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PMID:3',3",5',5"-Tetraiodophenolsulfonephthalein is a selective inhibitor of Ca(2+)-pumping ATPase in intracellular Ca2+ store. 802 Dec 63

Thrombin mitogenesis in fibroblasts requires two distinguishable subsets of signals; one generated by proteolytic cleavage, the other by high-affinity cell surface binding. Characterizing two closely related mouse embryo (ME) cell lines with high numbers of thrombin binding sites, we found that one line, B11-A, responds mitogenically to thrombin, epidermal growth factor (EGF), and serum, whereas the B11-B cell line is responsive to EGF and serum, but not to thrombin. The B11-B defect responsible for loss of thrombin responsiveness is not due to differences in the number of high-affinity binding sites, the affinity of thrombin binding to these sites, or to differences in cell surface expression of proteolytically activated receptors for thrombin (PART). The defect is also not associated with an inability of thrombin to activate PART since thrombin stimulates the cleavage-dependent induction of the proto-oncogene c-fos in both B11-A and B11-B cells. Various combinations of thrombin, synthetic thrombin receptor peptide, TRP-14 (SFFLRNPGENTFEL), platelet-derived growth factor (PDGF), and phorbol 12-myristate 13-acetate (PMA) were used to better define the defect in thrombin-mediated mitogenesis in B11-B cells. Direct activation of protein kinase C with PMA in combination with thrombin did not overcome B11-B nonresponsiveness. However, mitogenic responsiveness was regained in B11-B cells by simultaneous addition of PDGF and either thrombin or TRP-14. Therefore, the B11-B defect may involve a set of signals initiated by nonproteolytic thrombin interactions distinct from those initiated by PART, but related to the downstream signals initiated by the tyrosine kinase-associated growth factors, EGF and PDGF.
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PMID:Mouse fibroblasts defective in thrombin mitogenesis possess functional proteolytically activated receptor for thrombin: requirement for a second signaling pathway. 807 95

Astrocytes appear star-shaped in the brain, increasingly so after injury. When astroglia are cultured in serum-containing medium, they exhibit a flat, fibroblast-like morphology. In serum-free medium, astrocytes become stellate, with many long processes. The serine protease alpha-thrombin mimics the effects of serum at subnanomolar concentrations, whereas the thrombin-inhibiting serpin, protease nexin I (PNI), reverses the thrombin effect. In our current experiments, murine neonatal spinal cord astrocytes became stellate after 4 hr in serum-free medium, while cortical astrocytes required 12 hr in serum-free medium for stellation. Astrocytes from either region flattened after 60 min in medium containing 3.0 to 300 pM proteolytically active human alpha-thrombin. After 12 hr in thrombin-containing medium, 98% of the astrocytes had a flattened morphology. No flattening occurred if alpha-thrombin was replaced by gamma-thrombin, which has its fibrinogen-recognition exosite disrupted. PNI added at 1 nM to serum-containing medium caused stellation after 3 hr, and astroglia were 50% stellate by 12 hr. The effect of thrombin was mimicked by a 7-amino acid peptide (TRP-7) from the cleavage site of the human thrombin receptor. This peptide caused 40% of the astrocytes in serum-free medium to exhibit a flattened morphology after 6 hr. PNI had no effect on TRP-7 action on astrocytes. These results indicate that astrocytes possess a cell-surface receptor for thrombin, similar to that described for platelets, endothelial cells, and neurons.
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PMID:Thrombin receptor peptides induce shape change in neonatal murine astrocytes in culture. 814 98

A recently identified peptide sequence exposed after proteolytic cleavage of the NH2-terminus of the thrombin receptor mimics some cellular effects of alpha-thrombin. To determine whether a proteolytic action of thrombin is required for vasoactivity, we examined the vascular effects of modified thrombins and synthetic NH2-terminus peptide sequences of the thrombin receptor (TRPs) in isolated piglet lungs. Lungs of piglets 1-6 days old were perfused with recirculating Ringer-albumin solution at a constant flow of 60 ml/min. We measured the pulmonary artery pressure (Ppa) and segmental distribution of pulmonary vascular resistance (using the double occlusion method) in response to injections of human alpha-thrombin, modified thrombins, and TRP-14 and TRP-7 (i.e., 14 and 7 amino acid NH2-terminus peptides of the cleaved thrombin receptor). alpha-Thrombin produced a rapid and transient decrease in Ppa; the magnitude and duration [time for one-half recovery (t1/2 R)] of the vasodilation responses were concentration dependent [t1/2 R values of 1.4 +/- 0.1 and 3.3 +/- 2.4 min (mean +/- SE) at concentrations of 10(-10) and 10(-9) M, respectively]. The vasodilation was due primarily to a decrease in precapillary resistance. Proteolytically active, but binding-impaired gamma-thrombin was a less potent vasodilator and proteolytically inactive D-phenylalanyl-prolyl-arginine-chloromethyl ketone (PPACK)-alpha-thrombin did not induce vasodilation. TRP-14 was also a pulmonary vasodilator with a t1/2R value of 0.8 +/- 0.09 min at a concentration of 10(-7) M; both TRP-14 and TRP-7 were approximately 3-log less potent than equimolar alpha-thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Receptor mechanism of thrombin-mediated pulmonary vasodilation in neonates. 823 69

Thrombin, a potent activator of cellular responses, proteolytically cleaves, and thereby activates its receptor. In the present study, we compared the effects of the thrombin receptor 14-amino acid peptide (TRP-14; SFLLRNPNDKYEPF), which comprises the NH2 terminus after cleavage of the thrombin receptor, and of the native alpha-thrombin on endothelial monolayer permeability. Addition of TRP-14 (1-200 microM) to bovine pulmonary artery endothelial cells increased [Ca2+]i in a dose-dependent manner. The peak increase in [Ca2+]i in response to 100 microM TRP-14 or 0.1 microM alpha-thrombin was similar (i.e., 931 +/- 74 nM and 1032 +/- 80 nM, respectively), which was followed by a slow decrease with t1/2 values of 0.73 and 0.61 min, respectively. Extracellular Ca2+ chelation with 5 mM EGTA abolished the sustained increases in [Ca2+]i induced by either TRP-14 or alpha-thrombin. alpha-thrombin (0.1 microM) increased transendothelial [125I]albumin permeability, whereas TRP-14 (1-100 microM) had no effect. Coincubation of 100 microM TRP-14 with 1 microM DIP-alpha-thrombin also did not increase permeability over control values. Stimulation of BPAEC with 0.1 microM alpha-thrombin induced translocation of protein kinase C (PKC) from the cytosol to the plasma membrane indicative of PKC activation, whereas TRP-14 had no effect at any concentration. TRP-14 at 100 microM desensitized BPAEC to thrombin-induced increases in [Ca2+]i and transendothelial permeability. The Ca2+ desensitization was reversed after approximately 60 min, and this recovery paralleled the recovery of the permeability response. These findings indicate that the TRP-14-induced Ca2+ mobilization in the absence of PKC activation is insufficient to increase endothelial permeability. In contrast, the increase in endothelial permeability after alpha-thrombin occurred in conjunction with Ca2+ mobilization as well as PKC activation. TRP-14 pretreatment prevented the alpha-thrombin-induced increase in endothelial permeability secondary to desensitization of the Ca2+ signal. The results suggest that combined cytosolic Ca2+ mobilization mediated by TRP-14 and PKC activation mediated by a TRP-14-independent pathway are dual signals responsible for the thrombin-induced increase in vascular endothelial permeability.
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PMID:Thrombin receptor peptide inhibits thrombin-induced increase in endothelial permeability by receptor desensitization. 838 91

It has previously been reported that murine macrophages can respond chemotactically and mitogenically to the serine proteinase thrombin. There is a similar response in these macrophages to catalytically inactivated thrombin or to peptide fragments of the thrombin B-chain [Bar-Shavit, Kahn, Mann and Wilner (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 976-980]. However, the existence of a non-proteolytic mechanism of thrombin receptor activation in mononuclear cells was not evident in the present study using U937 human monocytic cells. The ability of thrombin to stimulate intracellular Ca2+ mobilization, actin polymerization or cell proliferation was not mimicked by N alpha-tosyl-L-lysine chloromethyl ketone (TLCK)-treated thrombin or by a synthetic 14-amino-acid peptide (single amino acid letter code YPPWNKNFTENDLL) corresponding to a part of the B-chain of thrombin which was reported to be mitogenic in murine macrophages. Evidence was obtained, however, in U937 cells for the presence of proteolytic-dependent thrombin receptor similar to the thrombin receptor expressed in platelets, which following thrombin cleavage exposes a new N-terminal tethered ligand. In support of this, a thrombin-receptor-derived hexapeptide (TRP; sequence SFLLRN), corresponding to a part of the thrombin receptor tethered ligand, mimicked all the actions of thrombin in U937 cells. Further, TRP and thrombin cross-desensitized U937 cells to subsequent stimulation with either TRP or thrombin, suggesting that TRP acted through the same U937 cell surface receptor as did thrombin. Thrombin activation of U937 monocytic cells can therefore be accounted for entirely by a proteolytic mechanism of thrombin receptor activation.
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PMID:The N-terminal thrombin receptor fragment SFLLRN, but not catalytically inactive thrombin-derived agonists, activate U937 human monocytic cells: evidence for receptor hydrolysis in thrombin-dependent signalling. 838 67

A working structural model of penicillin-binding protein 1B (PBP 1B) from Escherichia coli derived from previous data consists of a highly charged aminoterminal cytoplasmic tail, a 23-amino-acid hydrophobic transmembrane anchor, and a 758-amino-acid periplasmic domain. Using an engineered thrombin cleavage site, we have investigated the solubility properties of the periplasmic domain of PBP 1B. Twelve amino acids, comprised of the consensus thrombin cleavage site (LVPR decreases GS) and flanking glycine residues, were inserted into PBP 1B just past its putative transmembrane segment. To aid in purification, a hexa-histidine tag was also inserted at its amino terminus, and the engineered protein (PBP 1B-GT/H6) was purified and characterized. Inclusion of the thrombin cleavage site had no effect on the protein's intrinsic tryptophan fluorescence and affinity for [14C]penicillin G, indicating that the protein structure was not significantly perturbed. PBP 1B-GT/H6 was readily cleaved by thrombin at low thrombin/protein ratios to a protein with properties consistent with the removal of its cytoplasmic tail and transmembrane regions. Cleavage of the protein was dependent upon the presence of the thrombin cleavage site, and the thrombin-cleaved protein (PBP 1Bper) displayed an identical affinity for [14C] penicillin G binding as wild-type PBP 1B and uncleaved PBP 1B-GT/H6. [14C]Penicillin G-labeled PBP 1Bper eluted from a gel filtration column in the presence but not in the absence of 0.7% 3-[(3-cholamidopropyl)dimethylammonio]-1- propanesulfonic acid, and PBP 1Bper was found entirely in the membrane fraction of a thrombin digest of membranes containing overproduced PBP 1B-GT/H6. To further characterize this unusual solubility behavior, purified PBP 1Bper was reconstituted into lipid vesicles, which were then floated on a sucrose gradient. Floated vesicles contained > 95% of total 125I-penicillin V binding, indicating that PBP 1Bper directly associates with lipid membranes. These results strongly suggest that the periplasmic domain of PBP 1B associates with membranes independent of its amino terminal transmembrane region.
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PMID:Penicillin-binding protein 1B from Escherichia coli contains a membrane association site in addition to its transmembrane anchor. 844 26

Increases in intrinsic fluorescence (delta I), reflecting changes in tryptophan environments, occur upon bond cleavages necessary for prothrombin (II) activation to thrombin (IIa) by prothrombinase. Cleavage at Arg274-Thr275 (numbering based on bovine prothrombin sequence, with chymotrypsinogen numbering in braces) between the amino-terminal fragment 1.2 and protease (Pre2) domains of prothrombin yields delta I = 5%, and cleavage within the Pre2 domain at Arg323-Ile324 to form IIa yields delta I = 35%, while cleavage at both yields delta I = 25%. Since the change in fluorescence upon activation of prothrombin can be largely attributed to a change within the Pre2 domain, the susceptibilities of each of the 9 Trp residues of IIa and its immediate precursor Pre2 to oxidation by N-bromosuccinimide (NBS) were compared. Pre2 and IIa were titrated with increasing amounts of NBS (0.5-5 equiv of NBS/TRP), aliquots were removed and fully digested with trypsin, and tryptophan-containing peptides were separated and quantitated by RP-HPLC with fluorescence detection. Tryptic digests yielded 9 tryptophan-containing peptides, which were identified by amino acid composition. Tryptophan residues in IIa and Pre2 displayed a 10-fold range of sensitivity to modification. Tryptophans 337 and 360 (W29, W51) were modified less readily in IIa than in Pre2, while residues 373, 542, and 550 (W60D, W207, W215) were modified more readily, and other residues were equally susceptible. Residues 360 and 373 (W29, W60D) flank the active site histidine. From the crystal structure, residues 373 and 550 (W60D, W215) are implicated in substrate binding.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Structural changes in the protease domain of prothrombin upon activation as assessed by N-bromosuccinimide modification of tryptophan residues in prethrombin-2 and thrombin. 845 46

Subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex was over-expressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-RSIV) using the expression vector, pGEX/RSIV. Maximum yield of soluble active recombinant fusion protein was obtained from cells harvested 3 h after induction of growth at 37 degrees C in LB medium. Subunit IV was released from the fusion protein by proteolytic cleavage with thrombin. When subjected to SDS-polyacrylamide gel electrophoresis, isolated recombinant subunit IV of R. sphaeroides cytochrome b-c1 complex. Although the isolated recombinant subunit IV is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1000 kDa. The addition of detergent deaggregates the isolated protein, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. When the three-subunit core cytochrome b-c1 complex, purified from RS delta IV-adapted chromatophores containing a fraction of the wild type cytochrome b-c1 complex activity, was reacted with varying amounts of recombinant subunit IV, the activity increased as the subunit IV concentration increased. Maximum activity restoration was reached when 1 mol of subunit IV/mol of three-subunit core complex was used. The reconstituted cytochrome b-c1 complex is similar to the wild-type complex in molecular size, apparent Km for Q2H2, and inhibitor sensitivity, indicating that recombinant subunit IV is properly assembled into the active cytochrome b-c1 complex. A tryptophan residue in subunit IV was found to be involved in the interaction with the three-subunit core complex.
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PMID:Functional expression of subunit IV of Rhodobacter sphaeroides cytochrome b-c1 complex and reconstitution of recombinant protein with three-subunit core complex. 856 59


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