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)

A fraction separated from rat submandibular gland homogenates was found to contain a potent vasoconstrictor when tested on isolated rabbit aortic rings. The vasoconstrictor was purified by a series of chromatographic steps. The purified compound (2.77 x 10(-9) M) induced 40% of the maximum contractile response to 60 mM KCl. Constriction was slow in onset, long-lasting, rinse-resistant, and unchanged by de-endothelialization; in addition, it was dose-related and inhibited by both EGTA and verapamil, but it was not affected by DUP753, an angiotensin II receptor antagonist. The compound was found to be a protein having a pI of 7.36 and a molecular weight of approximately 29,000 and exhibiting partial immunologic identity to rat glandular kallikrein and rat tonin. After 2-mercaptoethanol treatment, it separated into heavy (approximately 19,900) and light (approximately 10,700) chains having amino-terminal sequences of AY(X)HNNDLMLL and VVGGYN(X)ETNSQ, respectively. We found that they correspond to the amino-terminal and internal sequence of a previously unidentified kallikrein-like serine protease whose mRNA, named S3, has been found in the rat submandibular gland and prostate. The vasoconstrictor is able to hydrolyze t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide (a thrombin substrate), although its Kcat/Km was only 0.02% that of rat thrombin. Both vasoconstrictor and enzymatic activity on t-butoxycarbonyl-valine-proline-arginine-methylcoumarin amide were completely suppressed by amidinophenylmethylsulfonyl fluoride and soybean trypsin inhibitor; however, they were unaffected by hirudin, a thrombin inhibitor. At pH 6.5, it released angiotensin II when incubated with sheep angiotensinogen, although it had approximately one-tenth the activity of tonin. The submandibular enzymatic vasoconstrictor is a kallikrein-like enzyme, having some properties of both tonin and thrombin. It directly contracts vascular smooth muscle, acting via a mechanism that requires intact enzymatic activity.
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PMID:A novel serine protease with vasoconstrictor activity coded by the kallikrein gene S3. 190 May 13

Antithrombin III Hamilton is a structural variant of antithrombin III (AT-III) with normal heparin affinity but impaired serine protease inhibitory activity. The molecular defect of AT-III-Hamilton is a substitution of threonine for alanine at amino acid residue 382. Recently it has been shown that both plasma-derived and cell-free-derived AT-III-Hamilton polypeptides act as substrates rather than inhibitors of thrombin and factor Xa. In the present study, the cell-free expression phagemid vector pGEM-3Zf(+)-AT-III1-432 was mutated at amino acid residue 382 of AT-III to generate 7 cell-free-derived variants. All these cell-free-derived AT-III variants were able to bind heparin as effectively as cell-free-derived normal AT-III. In terms of alpha-thrombin inhibitory activity each variant reacted differently. Variants could be grouped into 3 categories with respect to thrombin-AT-III complex formation: (1) near normal activity (glycine, isoleucine, leucine, valine); (2) low activity (threonine, glutamine); (3) no detectable activity (lysine). These data suggest that mutations at position 382 of AT-III may have a variable effect on protease inhibitory activity, depending on either the stability of the P12-P9 region of the exposed loop of AT-III, or the inability of the amino acid residue at position 382 to interact with a conserved hydrophobic pocket consisting of phenylalanine (at positions 77, 221 and 422) and isoleucine (position 412) residues.
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PMID:Site-directed mutagenesis of alanine-382 of human antithrombin III. 201 20

We have undertaken a structural and functional analysis of recombinant plasminogen activator inhibitor type 1 (PAI-1) produced in Escherichia coli using site-directed mutagenesis and immunochemistry. Expression of recombinant PAI-1 yielded an inhibitor that was functionally indistinguishable from PAI-1 made in human endothelial cells. Mutations in both the reactive center P1 and P1' residues (Arg-Met) and a putative secondary binding site for plasminogen activators on PAI-1 have been engineered to assess their functional effects. The inhibition of a panel of serine proteases, including plasminogen activators, trypsin, elastase, and thrombin, has been studied. Substitution of the P1 arginine residue with lysine or the P1' residue with either valine or serine had no detectable effect on the rate of inhibition of plasminogen activators. However, replacement of both P1 and P1' by Met-Ser produced a variant with no detectable plasminogen activator inhibitor activity. Mutations introduced into either Asp102 or Lys104 in the second site did not affect the rate of inhibition of plasminogen activators. Complementary immunochemical experiments using antibodies directed against the same two regions of the PAI-1 protein confirm that the reactive center is the primary determinant of inhibitory activity and that the putative second site is not a necessary functional region.
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PMID:Mutational and immunochemical analysis of plasminogen activator inhibitor 1. 212 Feb 33

Cerastobin, a thrombin-like enzyme, was isolated from the venom of Cerastes vipera (Sahara sand viper) in homogeneous form. Cerastobin had a molecular weight of 38,000 with 348 amino acid residues. It had an isoelectric point of 7.7 (a pH optimum of 7.9 and a temperature optimum of 45 degrees C). Cerastobin hydrolyzed arginine-containing synthetic substrates such as TAME, BAME, and BAEE, but BAPNA was not hydrolyzed. Cerastobin had thrombin-like activity, producing fibrin from fibrinogen and also hydrolyzing chromogenic substrates for thrombin such as 2AcOH.H-D-CHG-But-Arg-pNA (CBS 34.47) and H-D-Phe-Pip-Arg-pNA (S-2238). It showed kallikrein-like activity and hydrolyzed kallikrein substrates 2AcOH.H-D-Phe-Gly-Arg-pNA (CBS 33.27) and H-D-Pro-Phe-Arg-pNA (S-2302). It produced bradykinin from bradykininogen, as uterus contraction was observed. A serine inhibitor, DFP, exerted a pronounced inhibitory effect, suggesting that cerastobin is a serine-type protease. The sequence of 37 residues from the amino-terminal end was investigated. The amino-terminal amino acid was valine as it is in most other thrombin-like enzymes. The amino acid sequence of cerastobin was similar to that of thrombin in some residues and had some homology with that of kallikrein. However, cerastobin showed a high degree of homology to thrombin-like enzymes isolated from various snake venoms. Factor X was partially degraded by cerastobin. It was also found that antithrombin III was degraded by the enzyme. The alpha and beta chains of fibrin monomer were preferentially hydrolyzed by cerastobin, but the gamma chain was quite resistant.
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PMID:Characterization of cerastobin, a thrombin-like enzyme from the venom of Cerastes vipera (Sahara sand viper). 253 61

Hirudin is a 65-residue polypeptide that specifically inhibits thrombin by forming a tight, noncovalent complex with the enzyme. The role of the two amino-terminal valine residues and the N-terminal alpha-amino group of hirudin in the formation of the complex has been investigated by site-directed mutagenesis and chemical modification. Replacement of the two N-terminal valyl residues of recombinant hirudin by polar amino acids resulted in an increase in the inhibition constant (KI). In contrast, replacement of these residues by hydrophobic amino acids had little effect on the value for KI. These results demonstrated that the hydrophobic nature of the N-terminal residues of hirudin was important for its interaction with thrombin. Addition of a single amino acid to the N-terminus of hirudin resulted in a marked increase in the value of KI. A similar effect was observed when the positive charge of the alpha-amino group was removed by acetylation. In contrast, amidination of this group, which preserves the positive charge, resulted in a less pronounced increase in the value of KI. Thus, it appears that a positive charge immediately adjacent to the N-terminal hydrophobic residue is required for optimal binding to thrombin.
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PMID:Contribution of the N-terminal region of hirudin to its interaction with thrombin. 262 63

Human antithrombin III (AT) shares significant sequence homology and a common inhibitory mechanism with the serine protease inhibitor (serpin) superfamily. AT has a reactive site in which the P1 residue is primarily responsible for protease specificity. The P1' residue, almost invariably serine, is critical in the inactive natural variant AT-Denver, which has a leucine substitution in that position (Stephens, A.W., Thalley, B.S., and Hirs, C.H.W. (1987) J. Biol. Chem. 262, 1044-1048). In the present study site-directed mutagenesis was used to generate eight variants with altered P1' residues. All were secreted efficiently by COS cells transiently transfected with the AT cDNA in a eukaryotic shuttle vector. All variants also bound heparin as effectively as wild-type AT. Variants were grouped into three categories with respect to thrombin-AT complex formation: 1) no detectable inhibitory activity (proline, methionine); 2) low activity (cysteine, valine, leucine); and 3) near normal activity (glycine, alanine, threonine). The leucine variant, which is in the low activity group, exhibited the same physical and functional properties as AT-Denver. We conclude that the serine hydroxyl is not critical for functional activity and that there is a side chain size optimum which is modulated by hydrophobic effects.
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PMID:Site-directed mutagenesis of the reactive center (serine 394) of antithrombin III. 314 97

Determination of the nucleotide sequence of a cDNA for batroxobin, a thrombin-like enzyme from Bothrops atrox, moojeni venom, allowed elucidation of the complete amino acid sequence of batroxobin for the first time for a thrombin-like snake venom enzyme. The molecular weight of batroxobin is 25,503 (231 amino acids). The amino acid sequence of batroxobin exhibits significant homology with those of mammalian serine proteases (trypsin, pancreatic kallikrein, and thrombin), indicating that batroxobin is a member of the serine protease family. Based on this homology and enzymatic and chemical studies, the catalytic residues and disulfide bridges of batroxobin were deduced to be as follows: catalytic residues, His41, Asp86, and Ser178; and disulfide bridges, Cys7-Cys139, Cys26-Cys42, Cys74-Cys230, Cys118-Cys184, Cys150-Cys163, and Cys174-Cys199. The amino-terminal amino acid residue of batroxobin, valine, is preceded by 24 amino acids. This may indicate that the amino-terminal hydrophobic peptide (18 amino acids) is a prepeptide and that the hydrophilic peptide (6 amino acids), preceded by the putative prepeptide, is a propeptide.
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PMID:Molecular cloning and sequence analysis of cDNA for batroxobin, a thrombin-like snake venom enzyme. 354 2

Two arginine ester hydrolases, designated EI and EII, consist of multiple molecular forms with pI values in the range 4.0-4.6 for EI and 3.3-3.9 for EII. Isoforms had identical molecular weights: 38,500 for EI and 41,000 for EII (SDS electrophoresis). The N-terminal amino acid for both enzymes was valine and their amino acid contents were very similar, with both containing carbohydrate. After treatment of EI and EII with neuraminidase both enzymes migrated identically in the electrofocusing system. Neither esterase hydrolyzed casein, alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA), yet both hydrolyzed alpha-N-benzoyl-L-arginine methylester (BAEE), p-tosyl-L-arginine methylester (TAME) and Pro-Phe-Arg-MCA. The esterase activities of the two enzymes were inhibited by organophosphorus inhibitors and benzamidine. The Km value for EI with BAEE was 3.3 X 10(-5) M, with TAME 3.0 X 10(-5) M, and for EII 2.7 X 10(-5) M (BAEE) and 5.9 X 10(-5) M (TAME). EII possessed kinin-releasing activity, as shown by the twitch response of an isolated rat uterus. The physiological role of EI is unknown. Neither esterase has thrombin-like or fibrionlytic activities.
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PMID:Purification and characterization of two arginine ester hydrolases from Vipera berus berus (common viper) venom. 361 75

An abnormal fibrinogen was found in two asymptomatic members (father and daughter) of the same family, originating from northern Italy. Routine coagulation studies revealed prolonged thrombin and reptilase clotting times. Plasma fibrinogen levels, as determined by a functional assay, were markedly diminished, whereas the heat precipitation method indicated normal fibrinogen values. On the basis of these findings, a tentative diagnosis of dysfibrinogenemia was made, and according to the accepted nomenclature, this fibrinogen variant was called "fibrinogen Milano l." The time course of fibrinopeptide A and B release from fibrinogen Milano l was normal, but the aggregation of fibrin monomers was delayed. Two-dimensional electrophoresis of reduced variant fibrinogen chains showed a defective gamma-chain with increased cathodic mobility. An abnormal electrophoretic mobility was observed also for the gamma-chain remnants of fibrinogen fragments D1 and D2 derived from fibrinogen Milano l, whereas the charge anomaly was lost after a further digestion by plasmin to D3, suggesting that the structure abnormality of this variant is situated in the region gamma 303-356. An abnormal peptide was isolated after cyanogen bromide cleavage of intact fibrinogen Milano l. This fragment spans from position gamma 311 to gamma 336. Amino acid analysis of the abnormal peptide showed the presence of valine and a diminished content of aspartic acid. Sequence analysis demonstrated an amino acid exchange Asp----Val in the gamma-chain at position 330.
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PMID:Characterization of fibrinogen Milano I: amino acid exchange gamma 330 Asp----Val impairs fibrin polymerization. 370 59

A five-step isolation procedure has been developed for the purification of isoforms of hirudin (isohirudins) from whole leeches. The final purification of two thrombin-inhibiting preparations by reversed-phase high-performance liquid chromatography yielded several isohirudins with either N-terminal valine or isoleucine but with identical inhibition characteristics, i.e. specific thrombin inhibiting activities of 680-720 IU/mg and dissociation constants Ki of the thrombin-inhibitor complexes close to 3 X 10(-11) mol/l. The inhibitor with N-terminal isoleucine was designated hirudin PA. This inhibitor contains 66 amino-acid residues and has a molecular mass of 7,087 Da. The complete amino-acid sequence of hirudin PA was established by automated solid-phase Edman degradation of the native and oxidized inhibitor and two of its tryptic fragments. On the basis of the primary structures two types of thrombin inhibitors from the leech can be distinguished, designated hirudin and hirudin PA. The degree of structural homology of both isoinhibitors is approximately 82%; both have a tyrosine-O-sulfate residue near the C-terminus.
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PMID:Isolation and characterization of hirudin isoinhibitors and sequence analysis of hirudin PA. 376 44


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