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)

We have utilized circular dichroism spectroscopy to examine the interaction of antithrombin with heparin-derived oligosaccharides and mucopolysaccharides of various sizes. Our studies demonstrate that the various complexes exhibit two major types of chiral absorption spectra. The first of these patterns is seen when octasaccharide, decasaccharide, dodecasaccharide, or tetradecasaccharide fragments bind to the protease inhibitor. The circular dichroism spectra of these complexes when compared to the spectrum of free antithrombin show several distinguishing characteristics. On the one hand, there is a marked general increase in positive chiral absorption that is maximal at 296 and 288 nm and 290 and 282.5 nm. These observations indicate perturbation of "buried" and "exposed" tryptophan residues. On the other hand, a significant augmentation in circular dichroism that peaks at 269.5 and 263 nm is noted. These findings are probably due to the summed positive and negative contributions arising from tryptophan residue(s), disulfide bridge(s), and phenylalanine residue(s). Given that these heparin fragments are able to accelerate factor Xa-antithrombin interactions but not thrombin-antithrombin interactions, the above spectral transitions must be associated with either the binding of a critical domain of the oligosaccharides to the protease inhibitor or the "activation" of the protease inhibitor with respect to factor Xa neutralization. The second of these patterns is apparent when octadecasaccharide, low molecular weight heparin (6,500), and high molecular weight heparin (22,000) interact with antithrombin. The circular dichroism spectra of these complexes compared to the spectrum of free protease inhibitor are similar to the first pattern except for changes within the 292- to 282-nm and 275- to 255-nm regions. The subtraction of the first pattern from the second pattern reveals a shallow negative band between 300 and 275 nm with potential negative minima at 290 and 283 nm as well as a deep negative band between 275 and 255 nm with possible negative minima at 268 and 262 nm. This chiral absorption profile is most likely to arise from conformational changes of a disulfide bridge(s). However, we cannot completely exclude the possibility that the above circular dichroism difference curve might be explained on the basis of transitions originating from a tryptophan residue(s). Given our method for generating the above data, these spectral alterations must be associated with the binding of a second critical domain of the mucopolysaccharide to antithrombin that is required for rapid complex formation with thrombin or the activation of the protease inhibitor with respect to the neutralization of the latter enzyme.
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PMID:Circular dichroism spectroscopy of heparin-antithrombin interactions. 696 2

The effect of gamma-irradiation on purified prothrombin and thrombin in aqueous solution has been assessed with reference to bifunctional activities, e.g., clotting and esterase functions, physico-chemical changes in structure, and kinetics. The inactivation curves indicated that the clotting activity was more susceptible to gamma-radiation than the esterolytic function in both the proteins. Prothrombin was comparatively more sensitive to radiation than thrombin. The irradiation of prothrombin (100 kR) caused modifications in the protein resulting in reduced formation of thrombin after activation by Factor Xa. The modifications caused by irradiation were assessed in these proteins by changes in spectral characteristics, levels of tryptophan and disulphides, electrophoretic mobility and amino acid composition. Radiation-induced changes in thrombin were reflected in its kinetic behaviour. The clotting activity of thrombin was almost completely lost at 100 kR, while esterolysis was relatively less affected. The modification of tyrosine and tryptophan residues in thrombin influenced the clotting activity, while these were not involved for esterolysis. Histidine had involvement in both these activities.
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PMID:Radiation-induced changes in purified prothrombin and thrombin. 712 90

The interaction of alpha- and beta gamma-thrombin with heparin was studied by ultraviolet difference spectroscopy within the wavelength range of 230-300 nm. The absorption difference spectrum of the thrombin-heparin complex was negative and had two maxima at 255 nm (5300 M-1 cm-1) and 282 nm (4700 M-1 cm-1) for alpha-thrombin and at 240 nm (4900 M-1 cm-1) and 282 nm (4100 M-1 cm-1) for beta gamma-thrombin. It is assumed that the conformational changes induced by heparin in the enzyme molecule involve the transfer of some tryptophan and tyrosine residues from the interior of the protein to the surface. The absorption changes during alpha-thrombin--heparin interaction at physiological ionic strength suggest binding of some alpha-thrombin molecules to a heparin molecule at the ligand-enzyme molar ratio lower than 1. Under the same conditions beta gamma-thrombin forms an equimolar complex with heparin with the dissociation constant equal to 7,0.10(-9) M. The ionic strength increase up to 0,217 M NaCl results in some disturbances in beta gamma-thrombin-heparin interaction and prevents the binding of additional alpha-thrombin molecules to an equimolar complex of alpha-thrombin with heparin. Therefore the kinetics of the two enzyme forms interaction with heparin are similar, the alpha-thrombin affinity for heparin being a little higher. The data obtained suggest that alpha-thrombin binding to heparin is essential for biological inactivation of thrombin.
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PMID:[Ultraviolet difference spectroscopy study of alpha- and beta gamma-thrombin binding to heparin]. 724 53

The conformations of human alpha-thrombin and gamma-thrombin have been compared by circular dichroism, solvent perturbation different spectroscopy, and chemical modification. Circular dichroism studies indicate that proteolytic conversion of alpha-thrombin to gamma-thrombin is accompanied by considerable conformational changes which include a decrease in alpha-helical content from 5-7% to 0-1%. Solvent perturbation at pH 6.0 obtained with 20% ethylene glycol, 20% glycerol, and 20% dimethyl sulfoxide indicates an apparent exposure of 3.5 +2- 0.2 tryptophan and 7.8 +/- 0.1 tyrosine residues in alpha-thrombin and 4.6 +/- 0.2 tryptophan and 9.2 +/0 0.3 tyrosine residues in gamma-thrombin. This increased exposure is substantiated by the greater reactivity of tryptophan residues in gamma-thrombin toward dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide. It suggests that gamma-thrombin is a less compact molecule than the parent alpha-thrombin. Solvent perturbation studies of alpha-thrombin and gamma=thrombin inhibited by phenyl-methanesulfonyl fluoride showed that 0.3 +/- tryptophan and 0.9 +/- 0.3 tyrosine residues in alpha-thrombin and 0.6 +/- 0.3 tryptophan and 1.3 +/- 0.4 tyrosine residues in gamma-thrombin were blocked by the inhibitor. These subtle differences in the extent of blocking of tyrosine and tryptophan suggest a tighter conformation in the catalytic site of gamma-thrombin compared to that of alpha-thrombin.
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PMID:Conformational differences between high clotting human alpha-thrombin and nonclotting gamma-thrombin. 730 21

Chemical modification of antithrombin III, the major plasma protease inhibitor, with the tryptophan reagent dimethy(2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl moiety per molecule of antithrombin III. The derivatized inhibitor does not exhibit the heparin-promoted enhancement in rate of thrombin inactivation which is characteristic of the native molecule. However, the rates of thrombin inactivation in the absence of heparin are identical with native and derivatized inhibitors, indicating that the site of protease . inhibitor complex formation is not altered. Unlike native antithrombin III, the modified inhibitor does not bind to a heparin-agarose affinity column. When the modification reaction was performed with added heparin, the extent of modification was decreased and the heparin-promoted enhancement of thrombin inactivation was preserved. These results indicate that the integrity of a specific tryptophan residue is critical to the binding of heparin to antithrombin III.
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PMID:The heparin binding site of antithrombin III. Evidence for a critical tryptophan residue. 735 60

Heparin accelerates the rate of reaction of antithrombin with thrombin, an effect which is abolished by mild reduction of the antithrombin with dithiothreitol. Reduced antithrombin incorporates 1.7 mol of [14C]acetamide/mol of protein, with cysteine as the only amino acid modified. Tryptic digestion of the reduced and alkylated antithrombin results in the formation of only two labeled peptides. In the absence of heparin, the second order rate constant for the reaction of thrombin with both reduced and native antithrombin is 5.9 to 9.6 x 10(5) M-1 min-1. In the presence of heparin, the rate constant for the reaction between reduced antithrombin and thrombin is 8.3 to 12.2 x 10(5) M-1 min-1, while the rate of reaction between native antithrombin and thrombin is too fast to follow under the conditions used. Reduced antithrombin elutes from a heparin-Sepharose column at 0.5 M NaCl, contrast to 10 M NaCl required for elution of the native protein. The intrinsic tryptophan fluorescence enhancement caused by heparin binding to native antithrombin is not observed with reduced antithrombin. These data indicate that cleavage of one of the three antithrombin disulfide bonds results in reduced affinity for heparin and the loss of heparin-accelerated antithrombin activity and imply that heparin and thrombin bind at different sites on the antithrombin molecule.
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PMID:A disulfide bond in antithrombin is required for heparin-accelerated thrombin inactivation. 736 49

The activation of bovine prothrombin is known to be accompanied in purified systems by proteolytic reactions catalyzed by the product, thrombin. These reactions, which are directed principally towards the prothrombin substrate and the Factor V cofactor, are eliminated if the lysine residues of prothrombin are chemically modified beforehand with methyl acetimidate. Amidinated prothrombin in which the usual lysine content has been reduced by 75% is cleaved completely by Factor Xa to give thrombin which has little or no activity towards fibrinogen, the thrombin-sensitive bond in prothrombin, or Factor V, but with normal activity towards the synthetic chromogenic substrate D-Phe-Pipecolyl-L-Arg-p-nitroanilide. The formation of thrombin could therefore be studied spectrophotometrically by discontinuous assays of thrombin without complication by the proteolytic feedback activity of this enzyme. Such assays showed that the rate of appearance and yield of thrombin is the same whether from native or amidinated prothrombin, in spite of the lack of proteolytic activity in the product from the latter. Native and amidinated prothrombin have identical fluorescence emission spectra (lambda ex = 280 nm) which, upon activation, show a broadening and a red shift of the peak of emission from 330 to 336 nm. This change is different from the quenching known to occur when prothrombin binds Ca2+ and is contingent upon cleavage by Factor Xa. When monitored at the 370 nm band, the shift is seen as an increase in fluroescence intensity which, when the concentrations of Factor Xa and Factor V are adjusted appropriately, has the same general appearance as a progress curve obtained by discontinuous assay of thrombin activity. However, the curves obtained with the native zymogen appear to contain a component due to proteolysis by the accumulating product. in the case of amidinated zymogen, this is not longer so: the curves reflect only proteolysis by Factor Xa. In addition, a comparison of the fluorescence shift with the time course of thrombin appearance shows that the shift results mainly from the cleavage of intact prothrombin by Factor Xa, with little or no contribution from later events in the activation pathway. Modified Stern-Volmer plots for the quenching of fluorescence (lambda ex = 295 nm) of the intact amidinated zymogen and its activation products by sodium iodide allow the conclusion that activation results in the exposure to solvent of tryptophan residues that were previously sheltered. However, there is a variation in the pattern of quenching with the protein concentration, suggesting that these residues may be sheltered in the zymogen by intermolecular rather than intramolecular interactions.
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PMID:Chemically modified bovine prothrombin as a substrate in studies of activation kinetics and fluorescence changes during thrombin formation. 741 Mar 76

The cDNA encoding QPc-9.5 kDa (subunit VII) of bovine heart mitochondrial ubiquinol-cytochrome c reductase was cloned and sequenced. This cDNA is 665 base pairs long with an open reading frame of 246 base pairs that encodes an 81-amino acid mature QPc-9.5 kDa. The insert contains 395 base pairs of a 3'-noncoding sequence with a poly(A) tail. The amino acid sequence of QPc-9.5 kDa deduced from this nucleotide sequence is the same as that obtained by protein sequencing except that residue 61 is tryptophan instead of cysteine. The QPc-9.5 kDa was overexpressed in Escherichia coli JM109 cells as a glutathione S-transferase fusion protein (GST-QPc) using the expression vector, pGEX/QPc. The yield of soluble active recombinant GST-QPc fusion protein depends on the induction growth time, temperature, and medium. Maximum yield of recombinant fusion protein was obtained from cells harvested 3 h postinduction of growth at 27 degrees C on LB medium containing betaine and sorbitol. QPc-9.5 kDa was released from the fusion protein by proteolytic cleavage with thrombin. Isolated recombinant QPc-9.5 kDa showed one protein band in SDS-polyacrylamide gel electrophroesis corresponding to subunit VII of mitochondrial ubiquinol-cytochrome c reductase. Although the isolated recombinant QPc-9.5 kDa is soluble in aqueous solution, it is in a highly aggregated form, with an apparent molecular mass of over 1 million. Addition of detergent deaggreates the isolated protein to the monomeric state, suggesting that the recombinant protein exists as a hydrophobic aggregation in aqueous solution. The recombinant QPc-9.5 kDa binds ubiquinone and shows a spectral blue shift. Upon titration of the recombinant protein with ubiquinone, a saturation behavior is observed, suggesting that the binding is specific and that the recombinant protein may be in the functionally active state.
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PMID:Cloning, gene sequencing, and expression of the small molecular mass ubiquinone-binding protein of mitochondrial ubiquinol-cytochrome c reductase. 759 38

In an effort to clarify the role of GroES in chaperonin-facilitated protein folding, a plasmid-encoding expression system for GroES incorporating a histidine-tagged, thrombin-cleavable, N-terminal sequence was constructed. This approach facilitated the rapid purification of native-like, histidine-cleaved GroES (HC-GroES). The addition of NaSCN to purification buffers to mildly promote subunit dissociation enabled the complete separation of chromosomally encoded, wild-type GroES chains from recombinant chains, allowing the production of homogeneous mutant variants of GroES. A substitution of histidine-7 to tryptophan in GroES was used to demonstrate the concentration-dependent modulation of the heptameric quaternary structure of the chaperonin. Fluorescence and light scattering studies of this mutant suggest that GroES heptamers dissociate to monomers upon dilution with half-times of 2-4 min. Sedimentation equilibrium experiments using either wild-type or HC-GroES can best be described by a monomer--heptamer equilibrium, yielding dissociation constants of 1 x 10(-38) M6 for native GroES and 2 x 10(-32) M6 for HC-GroES. These results are supported by subunit exchange experiments using mixtures of native or HC-GroES and GroES containing the complete N-terminal histidine tail. Native polyacrylamide gel electrophoresis demonstrates that these mixtures form an eight-membered hybrid set within minutes. The studies described here suggest a dynamic equilibrium for the quaternary structure of GroES, which may be an important feature for its role in GroEL-mediated protein folding reactions.
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PMID:Monomer-heptamer equilibrium of the Escherichia coli chaperonin GroES. 765 86

Pleckstrin is a 40-kDa protein present in platelets and leukocytes that contains two PH domains separated by a 150-residue intervening sequence. Pleckstrin is a major substrate for protein kinase C, but its function is unknown. The present studies examine the effects of pleckstrin on second messenger generation. When expressed in cos-1 or HEK-293 cells, pleckstrin inhibited 1) the G alpha-mediated activation of phospholipase C beta initiated by thrombin, M1-muscarinic acetylcholine, and angiotensin II receptors, 2) the stimulation of phospholipase C beta by constitutively active Gq alpha, 3) the G beta gamma-mediated activation of phospholipase C beta caused by alpha 2A-adrenergic receptors, and 4) the tyrosine phosphorylation-mediated activation of phospholipase C gamma caused by Trk A. However, pleckstrin had no effect on either the stimulation or inhibition of adenylyl cyclase. The inhibition of phosphoinositide hydrolysis caused by pleckstrin was similar in magnitude to that caused by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and PMA had an additive effect, inhibiting phosphoinositide hydrolysis by as much as 90%. Structure-function analysis highlighted the role of pleckstrin's N-terminal PH domain in these events. Although deleting the C-terminal PH domain had no effect, deleting the N-terminal PH domain abolished activity (but not expression) and mutating a highly conserved tryptophan residue within the N-terminal PH domain decreased activity by one-third. Notably, however, a pleckstrin variant in which the N-terminal PH domain was replaced with a second copy of the C-terminal PH domain was nearly as active as native pleckstrin. These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase C beta- and phospholipase C gamma-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase C beta mediated by either G alpha or G beta gamma, but does not affect the regulation of adenylyl cyclase activity by G alpha or G beta gamma, 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckstrin and PMA are at least partially independent, 4) the inhibition caused by pleckstrin appears to be mediated by the PH domain at the N terminus, rather than the C terminus of the molecule, and 5) location of the two PH domains within the molecule clearly contributes to their individual activity.2+1
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PMID:Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains. 778 10


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