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)

The X-ray crystal structure of the serpin-proteinase complex suggested that the serpin deformed the proteinase thereby inactivating the molecule. Using a variant of alpha(1)-antitrypsin in which both tryptophan residues have been replaced by phenylalanine, we have shown that the proteinase becomes partially unfolded during serpin inhibition. The tryptophan free variant, alpha(1)-antitrypsin((FF)), is fully active as an inhibitor of thrombin. Thrombin has a fluorescence emission maximum of 340 nm which blue shifts to 346 nm, concomitant with a 40% increase in intensity, upon formation of the serpin-proteinase complex indicative of substantial conformational change within the proteinase. Stopped-flow analysis of the fluorescence changes within the proteinase indicated a two-step mechanism. A fast bimolecular reaction with a rate constant of 2.8x10(6) M(-1) s(-1) is followed by a slow unimolecular process with a rate of 0.26 s(-1) that is independent of concentration. We propose that the first rate is formation of an initial complex which is then followed by a slower process involving the partial unfolding of the proteinase during its translocation to the opposite pole of the serpin.
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PMID:Intrinsic fluorescence changes and rapid kinetics of proteinase deformation during serpin inhibition. 1129 29

Heparin has been proposed to conformationally activate the serpin, antithrombin, by making the reactive center loop P1 arginine residue accessible to proteinases. To evaluate this proposal, we determined the effect of mutating the P1 arginine on antithrombin's specificity for target and nontarget proteinases in both native and heparin-activated states of the serpin. As expected, mutation of the P1 arginine to tryptophan, histidine, leucine, and methionine converted the specificity of antithrombin from a trypsin inhibitor (k(assoc) = 2 x 10(5) M(-1) s(-1)) to a chymotrypsin inhibitor (k(assoc) = 10(3)-10(5) M(-1) s(-1)). However, heparin pentasaccharide activation increased the reactivity of the P1 variants with chymotrypsin or of the wild-type inhibitor with trypsin only 2-6-fold, implying that the P1 residue had similar accessibilities to these proteinases in native and activated states. Mutation of the P1 arginine greatly reduced k(assoc) for antithrombin inhibition of thrombin and factor Xa from 40- to 5000-fold, but heparin normally accelerated the reactions of the variant antithrombins with these enzymes to make them reasonably efficient inhibitors (k(assoc) = 10(3)-10(4) M(-1) s(-1)). Fluorescence difference spectra of wild-type and P1 tryptophan variant antithrombins showed that the P1 tryptophan exhibited fluorescence properties characteristic of a solvent-exposed residue which were insignificantly affected by heparin activation. Moreover, all P1 variant antithrombins bound heparin with approximately 2-3-fold higher affinities than the wild type. These findings are consistent with the P1 mutations disrupting a P1 arginine-serpin body interaction which stabilizes the native low-heparin affinity conformation, but suggest that this interaction is of low energy and unlikely to limit the accessibility of the P1 residue. Together, these findings suggest that the P1 arginine residue is similarly accessible to proteinases in both native and heparin-activated states of the serpin and contributes similarly to the specificity of antithrombin for thrombin and factor Xa in the two serpin conformational states. Consequently, determinants other than the P1 residue are responsible for enhancing the specificity of antithrombin for the two proteinases when activated by heparin.
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PMID:The antithrombin P1 residue is important for target proteinase specificity but not for heparin activation of the serpin. Characterization of P1 antithrombin variants with altered proteinase specificity but normal heparin activation. 1138 Feb 62

Room temperature tryptophan phosphorescence (RTTP) of human platelets in suspension was studied. The spectral and kinetic parameters of RTTP of thrombocytes were determined. By using the RTTP, the effect of serine proteases: thrombin, trypsin, and alpha-chymotrypsin at low concentrations (0-50 micrograms/ml) on the slow internal dynamics of membrane proteins was studied. It was shown that short-term incubation of platelets with proteases induces changes in the internal dynamics of membrane proteins in situ, and the magnitude of this effect correlates well with the extent of platelet aggregation. The functional role of the changes in the internal dynamics of membrane proteins and their relationship with intracellular signal transduction were discussed.
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PMID:[Effect of serine proteinases on the intracellular dynamics of membrane lipids and aggregation of human thrombocytes]. 1229 2

Snake venoms contain a large number of hemostatically active proteins that are structurally related to Ca(2+)-dependent animal lectins. These proteins, called C-type lectin-like proteins (CLPs), are generally found as heterodimers composed of two homologous subunits linked by a disulfide bond. Here, bothrojaracin (BJC), a CLP from Bothrops jararaca venom that is also a thrombin inhibitor, has been used as a model to study the subunit dissociation and unfolding of CLPs from snake venom. Dithiothreitol (DTT) up to 10 mM produces minor effects on the tertiary structure and activity of BJC. On the other hand, chromatographic studies and fluorescence polarization measurements indicate that the interchain disulfide bond is disrupted by DTT, although the dimeric association is maintained. Treatment of BJC with urea produces a progressive red shift in the emission spectra of the tryptophan residues, and circular dichroism measurements show that BJC retains significant secondary structure in the presence of 8 M urea, suggesting only partial unfolding. The effects of urea are fully reversible, as there is complete recovery of BJC activity after removal of the denaturing agent. Addition of DTT to a protein sample previously treated with 8 M urea produces a slightly larger spectral shift than that observed with urea alone. Furthermore, in this condition BJC loses its secondary structure, and its subunits are dissociated. After removal of urea and DTT, BJC is inactive toward thrombin, suggesting the irreversibility of their combined action. Altogether, our data show that (i) BJC is highly resistant to urea or DTT effects, requiring the simultaneous action of both agents to fully denature the protein, and (ii) BJC monomers are tightly associated, and the presence of DTT combined with high urea concentrations is necessary to disrupt them. On the basis of these results we propose the first denaturation model for a CLP from snake venom.
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PMID:Subunit dissociation, unfolding, and inactivation of bothrojaracin, a C-type lectin-like protein from snake venom. 1252 78

The P46 and P65 proteins of Mycoplasma hyopneumoniae are two membranous proteins carrying species-specific antigenic determinants. Based on the genomic sequence of the reference strain ATCC 25934, primers were designed for PCR amplification of the genes encoding entire P46 (1,260 bp) and P65 (1,803 bp) and N-terminally truncated P65(c) (1,200 bp). These primers were shown to be specific to M. hyopneumoniae since no DNA amplicons could be obtained with other mycoplasma species that commonly colonize the porcine respiratory tract. Both amplified genes were then cloned into the pGEX-4T-1 vector to be expressed in Escherichia coli cells as recombinant fusion proteins with glutathione S-transferase (GST). Prior to generation of expression constructs, TGA nonsense codons, exceptionally used for tryptophan residues by M. hyopneumoniae, had been converted to TGG codons by PCR-directed mutagenesis. Following induction by IPTG (isopropyl-beta-D-thiogalactopyranoside), both GST-P46 and GST-P65(c) recombinant fusion proteins were recovered by disrupting transformed cells by sonication, purified by affinity chromatography, and then cut with thrombin to release the P46 and P65(c) moieties. The enriched E. coli-expressed P46 and P65c proteins were used to immunize female BALB/c mice for the generation of anti-P46 and anti-P65(c) monoclonal antibodies (MAbs). The polypeptide specificities of MAbs obtained was confirmed by Western blotting with cell lysates prepared from the homologous strain. Cross-reactivity study of the anti-P46 and anti-P65(c) MAbs towards two other M. hyopneumoniae reference strains (ATCC 25095 and J strains) and Quebec field strains that had been isolated in culture, suggested that the MAbs obtained against both membranous proteins were directed against highly conserved species-specific epitopes. No reactivity to other mycoplasma species tested was demonstrated. Clinical signs and lesions suggestive of enzootic pneumonia were reproduced in specific-pathogen-free pigs that had been inoculated intratracheally with a virulent Quebec field strain (IAF-DM9827) of M. hyopneumoniae. Both anti-P46 and anti-P65(c) MAbs permitted effective detection by indirect immunofluorescence and indirect immunoperoxidase assay of M. hyopneumoniae in, respectively, frozen and formalin-fixed, paraffin-embedded lung sections from pigs that were killed after the 6- to 7-week observation period.
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PMID:Monoclonal antibodies to Escherichia coli-expressed P46 and P65 membranous proteins for specific immunodetection of Mycoplasma hyopneumoniae in lungs of infected pigs. 1273 49

Room temperature tryptophan phosphorescence (RTTP) of suspensions of human platelets was studied. RTTP spectra and decay kinetics of both intact platelets and those after short-term incubation with low concentrations of thrombin or trypsin (0.3-50 microg/ml) were investigated. Protease-induced changes in the RTTP lifetime of platelets were observed, and interpreted in terms of the slow internal dynamics of membrane protein modification. The functional role of membrane protein internal dynamics is discussed in the context of platelet aggregation and signal transduction processes.
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PMID:Slow internal dynamics of membrane proteins in mechanisms of protease-induced aggregation of platelets. 1284 97

Activation of bovine platelets with thrombin and phorbol 12,13-dibutyrate (PDBu) resulted in phosphorylation of profilin on serine. The phosphorylation was inhibited when platelets were pretreated with the PI 3-kinase inhibitor, LY294002, indicating that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation. Phosphorylation of profilin resulted in significant decrease in actin polymerization (16.5%), indicating an increased affinity of phosphoprofilin towards actin. The critical actin monomer concentration (Cc) increased to 260 nM in the presence of phosphoprofilin in comparison with 200 nM in the presence of profilin. The interaction of phosphoprofilin with phosphatidylinositol 4,5-bisphosphate [PI (4,5)-P2] and poly (L-proline) (PLP) was examined by monitoring the quenching of tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no difference in PI (4,5)-P2 binding between profilin and phosphoprofilin (Kd=20.4 microM), while poly (L-proline)-binding studies indicated a sixfold decrease (27.34 microM for profilin and 4.73 microM for phosphoprofilin) in Kd with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85alpha, the regulatory subunit of PI 3-kinase with phosphoprofilin over profilin. Overall, the data presented conclude that profilin phosphorylated under in vivo conditions and phosphorylation depends upon activation of PI 3-kinase. Phosphoprofilin exhibited increased affinity to poly (L-proline) sequences both in vitro and in vivo.
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PMID:Phosphorylation of profilin regulates its interaction with actin and poly (L-proline). 1475 44

7-Azatryptophan (AW), a noncoded isostere of tryptophan (W), possesses interesting spectral properties. In particular, the presence of a nitrogen atom at position 7 in the indolyl nucleus of AW results in a red shift of the absorption maximum and fluorescence emission by 10 and 46 nm, respectively, compared to W. In the present work, we report the chemical synthesis and the conformational and functional characterization of an analog (denoted as Y3AW) of the N-terminal domain 1-47 of hirudin, a highly potent thrombin inhibitor, in which Tyr 3 has been replaced by AW. The results obtained were compared with those of the corresponding Y3W analog. We found that the replacement W --> AW reduces affinity for thrombin by 10-fold, likely because of the lower hydrophobicity of AW compared with that of W. Measurements of the resonance energy transfer effect, which was observed between Tyr13 and the amino acid at position 3 upon disulfide-coupled folding, demonstrate that AW behaves as a better energy acceptor than W for studying protein renaturation. The interaction of Y3AW with thrombin was studied by exciting the sample at 320 nm and recording the change in fluorescence of Y3AW on binding to the enzyme. Our results indicate that the fluorescence of AW of hirudin 1-47 in the Y3AW-thrombin complex is strongly quenched, possibly because of the presence of two structural water molecules at the hirudin-thrombin interface that can promote the nonradiative decay of AW in the excited state. The data herein reported demonstrate that the incorporation of AW can be of broad applicability in the study of protein folding and protein-protein interaction.
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PMID:Incorporation of the fluorescent amino acid 7-azatryptophan into the core domain 1-47 of hirudin as a probe of hirudin folding and thrombin recognition. 1515 84

Mycoplasma hyopneumoniae is the most significant bacterial pathogen of the respiratory tract of swine. p65 is an immunodominant surface lipoprotein of M. hyopneumoniae that is specifically recognized during disease. Analysis of the translated amino acid sequence of the gene encoding p65 revealed similarity to the GDSL family of lipolytic enzymes. To examine the lipolytic activity of p65, the gene was cloned and expressed in Escherichia coli after truncation of the prokaryotic lipoprotein signal sequence and mutagenesis of the mycoplasma TGA tryptophan codons. After treatment with thrombin, the recombinant glutathione S-transferase (GST)-p65 protein yielded a 66-kDa fusion protein cleavage product corresponding in size to the mature p65 protein. The esterase activity of recombinant GST-p65 was indicated by the formation of a cleared zone on tributyrin agar plates and the hydrolysis of p-nitrophenyl esters of caproate (pNPC) and p-nitrophenyl esters of palmitate (pNPP). Lipase activity was indicated by the hydrolysis of the artificial triglyceride 1,2-O-dilauryl-rac-glycero-3-glutaric acid resorufin ester. Using pNPC and pNPP as substrates, recombinant GST-p65 had optimal activity between pHs 9.2 and 10.2 and at a temperature higher than 39 degrees C. Calcium ions did not increase the activity of recombinant GST-p65. Rabbit anti-p65 antibodies inhibited the activity of recombinant GST-p65 and also inhibited the growth of M. hyopneumoniae in vitro. Examination of the kinetic parameters of recombinant GST-p65 for the hydrolysis of pNPC and pNPP indicated a preference for the shorter fatty acid chain of pNPC. The physiological and/or pathogenic role of mycoplasma lipolytic enzymes has not been determined, but they are likely to play an important role in mycoplasmas' nutritional requirements for long-chain fatty acids and may reduce the function of lung surfactants in mycoplasma-induced respiratory diseases. This is the first report of the lipolytic activity of a lipid-modified surface immunogen of a mycoplasma.
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PMID:Mycoplasma hyopneumoniae p65 surface lipoprotein is a lipolytic enzyme with a preference for shorter-chain fatty acids. 1531 84

The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB (the TolB box). Previous NMR studies (Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D (1)H-(13)C-(15)N NMR experiments, and the backbone dynamics of these regions investigated through measurement of (1)H-(15)N relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R(2) relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain. Abbreviations : Im9, the immunity protein for colicin E9; E9 DNase, the endonuclease domain of colicin E9; HSQC, heteronuclear single quantum coherence; ppm, parts per million; DSS, 2,2-(dimethylsilyl)propanesulfonic acid; TSP, sodium 3-trimethylsilypropionate; T(1 - 61)-DNase fusion protein, residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight residue thrombin cleavage sequence.
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PMID:Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9. 1545 37


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