Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.86 (clotting enzyme)
 176 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine prothrombin was activated, in both the absence and presence of dissopropyphosphofluoridate (DEP) and benzamidine, by an activator which was highly purified from the venom of Echis carinatus (saw-scaled viper, ECV). The process of activation was monitored by sodium dodecysulfate (SDS)-polyacrylamide gel electrophoresis, and the reaction products were isolated and chemically characterized. In the absence of the inhibitors, prothrombin yielded two fragments with molecular weights of 28,000 and 57,000, of which the former was the N-terminal fragment of the zymogen and the latter was intermediate 1, consisting of a single polypeptide chain. Intermediate 1 was subsequently converted to an active intermediate, named intermediate ECV, without decrease of molecular weight. This new intermediate ECV, which showed little clotting activity but a strong alpha-N-tosyl-L-arginine methyl ester (TAME)-esterolytic activity and which bound with hirudin or antithrombin III, consisted of two polypeptide chains with molecular weights of 35,000 of 27,000 daltons. The former was indentified as the thrombin B chain with the N-terminal sequence Ile-Val-Glu-Gly and C-terminal serine, and the latter was a fragment with N-terminal Ser-Gly-Gly, linked to the thrombin A chain. On prolonged incubation, intermediate ECV autocaralytically yielded a fragment (inner fragment) of 14,000 daltons with N-terminal serine and the clotting enzyme alpha-thrombin [EC 3.4.21.5], which consists of A and B chains. In the presence of the inhibitors, intermediate ECV and the N-terminal fragment were accumulated in the activation mixture. On the other hand, when prothrombin was activated by the venom activator in the presence of hirudin, antithrombin III, or p-nitrophenyl p'-guanidinobenzoate, it did not yield any fragments but was converted to a derivative with two polypeptide chains having molecular weights of 51,000 and 34,000 daltons, of which the former consisted of N-terminal fragment, the inner fragment, and thrombin A chain, and the latter was thrombin B chain. This new prothrombin derivative, named prothrombin ECV, formed a high-molecular-weight complex, associating with antithrombin III. The complex was not dissociable even in the presence of SDS. Moreover, prothrombin ECV reacted with p-nitrophenyl p'-guanidinobenzoate. On the basis of the results described above, the mechanism of activaton of prothrombin by Echis carinatus venom activator can be summarized as follows: The venom activator first cleaves an Arg-Ile bond liniking thrombin A and B chains in the zymogen molecule, forming an active derivative, prothrombin ECV. This active derivative converts autocatalytically to intermediate ECV, liberating the N-terminal fragment, and active intermediate ECV generates alpha-thrombin, releasing the inner fragment. Thus, only a single peptide bond cleavage along the polypeptide chain of prothrombin is associated with activation by the venom activator...
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PMID:The mechanism of activation of bovine prothrombin by an activator isolated from Echis carinatus venon and characterization of the new active intermediates. 95 36

A clottable protein (coagulogen) isolated from a hemocyte lysate of the Japanese horseshoe crab (Tachypleus tridentatus) was incubated with an endotoxin-activated clotting enzyme(s) partially purified from the same lysate, and its structural change during gel formation was examined. The results indicated that the enzymatic formation of gel involved limited proteolysis of the Arg-Gly and Arg-Thr linkages located in the N-terminal portion of the coagulogen, liberating peptide C.
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PMID:A clottable protein (coagulogen) of horseshoe crab hemocytes. Structural change of its polypeptide chain during gel formation. 97 58

A clotting enzyme, associated with the endotoxin-mediated activation of the cellularly based coagulation system of the American horseshoe crab (Limulus polyphemus), was considerably purified by a modification of the method employed to purify the corresponding enzyme from the Japanese horseshoe crab (Tachypleus tridentatus) (Nakamura, et al., 1982). This enzyme was inhibited by DFP, benzamidine, p-aminobenzamidine, antithrombin III, soybean trypsin inhibitor, and antipain, suggesting that it is a trypsin-type serine protease. The enzyme demonstrated amidolytic activity to Ac-Ile-Glu-Gly-Arg-pNA (S-2423) and related synthetic substrates (S-2222, S-2422, S-2337, and Boc-Leu-Gly-Arg-pNA) but not to other substrates (S-2160, S-2238, S-2251, S-2444, S-2266, and S-2302), indicating specificity similar to mammalian blood coagulation Factor Xa. These properties of the Limulus enzyme were identical with those of the corresponding Tachypleus enzyme. The structure and function of the enzymes in these two species probably have been highly conserved during the past few hundred million years of their evolution.
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PMID:Properties of the clotting enzyme responsible for endotoxin-mediated Limulus coagulation. 637 72

The complete amino acid sequence of the coagulogen from hemocyte lysates of Limulus polyphemus has been determined by sequencing the peptides obtained from tryptic, chymotryptic, staphylococcal protease V8 and lysyl endopeptidase digestions. These results established the following sequence: (formula; see text) Limulus coagulogen consists of a single chain with a total of 175 amino acid residues and the molecular weight is calculated to be 19,675. It contains 16 half-cystines in disulfide linkages, with 5 half-cystines located in a cluster in the COOH-terminal 14 residues. The sequence of Limulus coagulogen is very close to that for the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab), having 69% sequence homology. The 16 half-cystines of these coagulogens are in the same positions, suggesting a very similar conformation. Moreover, the COOH-terminal tripeptide regions of the A chain (from the NH2-terminal end to Arg-18) and peptide C (from Lys-19 to Arg-46), both of which seem to interact with a Limulus clotting enzyme to liberate peptide C, are completely conserved. From secondary structure predictions by the method of Chou and Fasman (Chow, P.Y., and Fasman, G. D. (1974) Biochemistry 13, 211-222), the coagulogen appears to contain an alpha-helical region in the peptide C segment, released by the clotting enzyme, suggesting a marked conformational change in the transformation of the coagulogen to the coagulin gel. beta-sheet and reverse turn regions are distributed in the B chain segment (from Gly-47 to the COOH-terminal end). It is likely that the 16 half-cystines and abundant beta-sheet structure make the coagulogen molecule compact.
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PMID:Amino acid sequence of the coagulogen from Limulus polyphemus hemocytes. 637 4

A clotting enzyme associated with the hemolymph coagulation system of Japanese horseshoe crab (Tachypleus tridentatus) was highly purified from the amebocyte lysate. The method for purification consisted of gel-filtration of the lysate on a pyrogen-free Sepharose CL-6B column and affinity chromatography of the endotoxin-treated clotting enzyme on a benzamidine-Sepharose 4B column. Through these procedures, about 3 mg of the purified enzyme was obtained from 70 ml of the lysate and about 390-fold purification was achieved. The purified preparation was found to give a single major band, respectively, on polyacrylamide-gel disc electrophoresis at pH 3.2 in the presence of 6 M urea and on sodium dodecyl sulfate (SDS)-gel electrophoresis in the presence and absence of 2-mercaptoethanol. It also gave a single symmetrical peak on QAE-Sephadex A-25 column chromatography. The molecular weight of the clotting enzyme was estimated to be approximately 42,000 for the unreduced sample by SDS-gel electrophoresis. For the reduced sample, it was 30,000, suggesting that the protein consists of plural polypeptide chains bridged by disulfide(s). The Tachypleus clotting enzyme was a glycoprotein, as shown by the positive periodic acid-Schiff reaction for the protein band on SDS-gel and the amino acid analysis. The purified clotting enzyme transformed Tachypleus coagulogen to coagulin gel and hydrolyzed a chromogenic peptide substrate, Tos-Ile-Glu-Gly-Arg-p-nitroanilide for Factor Xa, liberating p-nitroaniline. The enzyme was sensitive to DFP and benzamidine. It was also inhibited partially by PCMB. Antithrombin III and alpha 2-plasmin inhibitor (alpha 2-antiplasmin) were very effective inhibitors of this enzyme among ten kinds of naturally occurring proteinase inhibitors tested. The clotting enzyme had a restricted specificity towards protein substrates and activated only prothrombin among plasma zymogens including Factor IX, Factor X, fibrinogen, plasminogen and prekallikrein. The cleavage sites of bovine prothrombin for this enzyme were the same Arg-Thr and Arg-Ile linkages as those for Factor Xa, resulting in the formation of alpha-thrombin. These results indicate that the horseshoe crab clotting enzyme is a Factor Xa-like serine proteinase rather than alpha-thrombin. It seems likely that the Tachypleus clotting enzyme is a prototype of mammalian serine proteinases participating in blood coagulation.
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PMID:A clotting enzyme associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus): its purification and characterization. 714 17

Lipoglycans (previously designated lipopolysaccharides) from several species of Acholeplasma and from Thermoplasma acidophilum were examined for endotoxin-like activities as measured by the standard rabbit fever test and the Limulus amoebocyte lysate assay. The lipoglycans from Acholeplasma granularum, Achloplasma laidlawii, Acholeplasma modicum, and Acholeplasma oculi caused a febrile response at concentrations of 1 ng/ml per kg or greater, whereas with control Escherichia coli EC-2 lipopolysaccharides, 6.25 ng/ml per kg was required. Similar results were obtained in the Limulus amoebocyte lysate test. The minimum concentrations in nanograms per milliliter required to stimulate formation of a solid clot were: Acholeplasma axanthum, 0.22; A. granularum, 0.85; A. modicum, 0.51; A. laidlawii, 1.05; A. oculi, 0.74. Standard E. coli 1B lipopolysaccharide required a concentration of 0.125 ng/ml. Thermoplasma lipoglycan was least active, requiring 4.25 ng/ml. Clotting of the Limulus lysate proceeds by the activation by lipopolysaccharide plus Ca(2+) of a proenzyme which cleaves an arginine-lysine peptide bond of the coagulogen. The clotting and amidase activities are inactivated by deoxycholate and can be reactivated by addition of lipopolysaccharide and Ca(2+). As with E. coli 1B lipopolysaccharide, acholeplasmal lipoglycans were shown to restore both clotting and amidase activities of the deoxycholate-inactivated Limulus clotting enzyme. The degree of restoration of amidase activity by mycoplasmal lipoglycans relative to E. coli lipopolysaccharide (1.00) were: A. axanthum, 1.71; A. modicum, 1.22; A. granularum, 0.61; and Thermoplasma, 0.37. The coagulating enzyme, restored with either E. coli lipopolysaccharide or mycoplasmal lipoglycans, was able to react with the synthetic peptide benzoyl-Ile-Glu-(gamma-OCH(3))-Gly-p-nitroaniline (an analog of the coagulogen) or with the purified coagulogen itself to form the clot. The mycoplasmal lipoglycans alone were incapable of promoting these reactions when incubated with the synthetic peptide or with the purified coagulogen, thereby ruling out the contamination of these lipoglycans with proteases capable of cleaving the same Arg-Lys peptide bond of the coagulogen. These results show that acholeplasmal lipoglycans possess endotoxin-like activities. Their passive or active role in disease remains to be established.
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PMID:Endotoxin-like activities of mycoplasmal lipopolysaccharides (lipoglycans). 742 42

Moulting fluid of pharate adult tobacco hornworm moths, Manduca sexta, contains a novel cuticle-degrading proteinase, designated as MFP-1. The enzyme has been purified using heparin affinity chromatography and partially characterized. Before purification MFP-1 is associated with a large complex having an apparent native molecular mass > 669 kDa. After purification MFP-1 has a molecular mass of 41 kDa. The pI of the enzyme is 5.54. MFP-1 can be classified as generally trypsin-like on the basis of its substrate specificity and inhibition by soybean trypsin inhibitor. The enzyme's preferred substrate, Tos-Gly-Pro-Arg-pNA, its inhibition by hirudin, and its affinity for heparin, all indicate that MFP-1 has some characteristics in common with the vertebrate blood-clotting enzyme thrombin. MFP-1 is probably a serine protease, since it is inhibited by both DFP and PMSF (specific inhibitors of serine proteinases). However, the enzyme was also inhibited by a number of agents that affect cysteine proteinases. Purified MFP-1 degrades Manduca cuticle in vitro. We suggest that the enzyme may act as the first step in the degradation of the cuticle during the moulting process.
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PMID:A cuticle-degrading proteinase from the moulting fluid of the tobacco hornworm, Manduca sexta. 835 21

A novel (1-->3)-beta-D-glucan-binding protein (T-GBP) has been purified from the amoebocyte lysate of the Japanese horseshoe crab, Tachypleus tridentatus. It is a basic protein (pI 9.2) which appears to be a homotetramer composed of subunits with an apparent mol wt of 168000 and with an amino-terminal sequence (20 residues) KSGFILTAPKSLTLGRNNRL. T-GBP exerted an inhibitory effect on the (1-->3)-beta-D-glucan-initiated coagulation cascade reconstituted with purified preparations of factor G and the proclotting enzyme from the lysate. The binding of (1-->3)-beta-D-glucans to T-GBP was evaluated by measuring the residual amidolytic activity of the clotting enzyme, the product of the coagulation cascade, using Boc-Leu-Gly-Arg-4-nitroanilide as the chromogenic substrate. The binding specificity of a wide range of (1-->3)-beta-D-glucans and other polysaccharides towards T-GBP was expressed by the relative inhibition (%) of the activation of factor G, the first protease zymogen in the pathway, which is activated by binding to (1-->3)-beta-D-glucans. T-GBP was found to have a high affinity for linear (1-->3)-beta-D-glucans, e.g. pachyman, curdlan, and paramylon. It was able to bind to (1-->3)-beta-D-glucans with side-chain branches and mixed linkage such as schizophyllan, lentinan, laminarins, yeast beta-D-glucan, and (1-->3),(1-->4)-beta-D-glucans such as lichenin and barley beta-D-glucan. Binding of pachyman to T-GBP was demonstrated by an enzyme-linked immunosorbent assay using a specific antibody (rabbit IgG) raised against T-GBP.
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PMID:Purification and characterization of a (1-->3)-beta-D-glucan-binding protein from horseshoe crab (Tachypleus tridentatus) amoebocytes. 900 87

A new endogenous serine proteinase from the cell-free hemolymph of a solitary ascidian, Halocythia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on TSKgel Toyopearl HW 65 F, ion exchange chromatography on TSKgel DEAE-Toyopearl 650 M, affinity chromatography on Arginine-Sepharose 4B, gel filtration on TSKgel Toyopearl HW 65F and hydroxyapatite chromatography on Bio-Gel HT. The serine proteinase is a single polypeptide chain whose molecular weight and isoelectric point are 39 kDa and about 7.6 pI, respectively. The most susceptible substrate was Boc-Leu-Gly-Arg-4-methyl-coumaryl-7-amide (MCA), and activity was optimal at pH 8. The enzyme was relatively stable at high temperatures; about 50% activity was retained even at 60 degrees C for 30 min in 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and 0.05% Brij-35. The enzyme was characterized by the inhibitory effects of synthetic or natural inhibitors, substrate specificity toward 26 peptidyl-MCAs, proteinase activity toward natural proteins and complex formation with a serine proteinase inhibitor (58 kDa) previously found in H. roretzi hemolymph, indicating that the enzyme was a member of serine proteinases and strongly inhibited by the 58 kDa serine proteinase inhibitor as well as human antithrombin III. We also demonstrated the clotting enzyme activity of the purified serine proteinase toward bovine fibrinogen and Limulus coagulogen, a fibrinogen-like clottable protein of horseshoe crabs.
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PMID:Purification and characterization of a 39,000-Da serine proteinase from the hemolymph of a solitary ascidian, Halocynthia roretzi. 941 2

The structure of two selective inhibitors, Ac-Tyr-Ile-Arg-Ile-Pro-NH2 and Ac-(4-Amino-Phe)-(Cyclohexyl-Gly)-Arg-NH2, in the active site of the blood clotting enzyme factor Xa was determined by using transferred nuclear Overhauser effect nuclear magnetic resonance (NMR) spectroscopy. They represent a family of peptidic inhibitors obtained by the screening of a vast combinatorial library. Each structure was first calculated by using standard computational procedures (distance geometry, simulated annealing, energy minimization) and then further refined by systematic search of the conformation of the inhibitor docked in the active site and repeating the simulated annealing and energy minimization. The final structure was optimized by molecular dynamics simulations of the inhibitor-complex in water. The NMR restraints were kept throughout the refinement. The inhibitors assume a compact, very well defined conformation, embedded into the substrate binding site not in the same way as a substrate, blocking thus the catalysis. The model allows to explain the mode of action, affinity, and specificity of the peptides and to map the active site.
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PMID:Mapping the active site of factor Xa by selective inhibitors: an NMR and MD study. 951 42


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