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)

A pro-clotting enzyme capable of causing the gelation of clottable proteins in Limulus polyphemus (horseshoe crab) has been purified to apparent homogeneity as judged by sodium dodecyl sulfate-gel electrophoresis. The activation of the pro-clotting enzyme depended on the presence of both Ca+ and endotoxin. It contained gamma-carboxyglutamic acids and gave a single NH2-terminal lysine. The enzyme was inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, and soy bean trypsin inhibitor, indicating that it is a serine protease. The molecular weight of the proclotting enzyme was determined to be at least 150,000 by sodium dodecyl sulfate-gel electrophoresis under reducing and denaturing conditions. The protein appears to consist of a single peptide chain, since exposure of the reduced and carboxymethylated enzyme to 6 M guanidine hydrochloride failed to dissociate it into any subunits.
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PMID:Studies on Limulus amoebocyte lysate. Isolation of pro-clotting enzyme. 32 51

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.
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PMID:Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization. 226 34

A fibrinogen-clotting enzyme from the venom of the Peruvian bushmaster snake was purified to homogeneity by gel filtration on Sephadex G-100 followed by DEAE-cellulose ion-exchange chromatography using a linear ionic strength gradient with NaCl. The specific activity of the enzyme was 866 NIH U/mg, representing a 55-fold purification, with a recovery of 45%. The amino acid composition was Asx30, Thr14, Ser15, Glx33, Pro23, Gly22, Ala15, Val22, Cys18, Met3, Ile18, Leu23, Tyr2, Phe13, His8, Lys11, Arg11. The total carbohydrate content was 13.4%, comprised of 3.4% hexose, 8.7% hexosamine and 1.3% sialic acid. The enzyme was active against the synthetic amide substrate alpha-N-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and against the ester substrates alpha-N-benzoyl-L-arginine ethyl ester (BAEE) and tosyl-L-arginine methyl ester (TAME). Kinetic parameters for TAME esterolysis were: Vmax, 135 mumoles/min/mg and Km, 2.5 x 10(-4) M. The pH optimum was 8.0. Vmax for BAPNA amidolysis was 0.363 mumoles/min/mg and Km, 7.5 x 10(-5) M. Enzyme activity was reduced by diethylpyrocarbonate and by photo-oxidation, suggesting that the enzyme is a serine protease with a histidine residue involved in the active site. The enzyme released fibrinopeptide A rapidly from purified human fibrinogen and fibrinopeptide B more slowly. Factor XIII was not activated and the clotting activity was not inhibited by heparin. A dose of 50 micrograms/kg brought about defibrinogenation in anaesthetized rats but rabbits were unaffected. A dose of 80 micrograms/kg defibrinogenated conscious rats after 5 hr. There were no hypotensive or haemorrhagic effects.
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PMID:Isolation and characterization of a fibrinogen-clotting enzyme from venom of the snake, Lachesis muta muta (Peruvian bushmaster). 261 37

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 blood coagulation protein factor Va forms the receptor for the serine protease factor Xa on the platelet surface. This membrane-bound complex of factor Va and factor Xa plus Ca2+ comprises the prothrombinase complex, the enzyme that catalyzes the proteolytic conversion of prothrombin to the clotting enzyme thrombin. Factor Va is a two-subunit protein composed of component D (Mr = 94,000) and component E (Mr = 74,000); subunit interaction is Ca2+ dependent. Factor Va bound to platelets consists of three peptides: component D, component E, and component D'(Mr = 90,000) which appears as the result of a platelet-associated protease cleavage of component D. The present studies were undertaken to determine which peptide(s) mediates the binding of factor Va to the platelet membrane surface and which peptide(s) serves as the binding site for factor Xa. These interactions were assessed by direct measurements of radiolabeled factor Va and factor Xa binding to platelets as well as autoradiographic visualization of the factor Va peptides associated with the platelet. Experiments were performed to determine the interaction of components D and E with platelets under reaction conditions in which components D and E were present as either the intact, functional two-subunit protein or as nonfunctional discrete peptides dissociated by the addition of Na2EDTA. The results suggest that component E mediates the binding of factor Va to the platelet and also serves as the binding site for the interaction of factor Xa with platelet-bound factor Va.
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PMID:Prothrombinase complex assembly on the platelet surface is mediated through the 74,000-dalton component of factor Va. 657 81

Prothrombinase is a Ca2+-dependent, 1:1, enzymatic complex of Factor Xa and Factor Va that assembles on the surface of negatively charged phospholipid vesicles or platelets. It catalyzes the proteolytic conversion of prothrombin to the blood-clotting enzyme thrombin. Experimentally determined kinetic parameters, plus Kd and n values for the interaction of substrate, cofactor (Factor Va), and serine protease (Factor Xa) for both phospholipid and each other, were used to develop a model that simulates the functional properties of the enzymatic complex. Through the use of a desk-top computer and a program designated "Clotspeed," the distribution of enzymatic components and substrate between the bulk fluid and phospholipid is determined for a given set of initial concentrations of reaction components. Simulated reaction rates are then calculated from the calculated distributions, fractional binding, and local and bulk concentration of reactants. Predicted behavior includes formal Michaelis-Mentenlike properties for the reaction, increasing apparent Km with increased levels of phospholipid, and apparent inhibition by excess substrate, enzyme, and phospholipid. Inhibition by excess enzyme and phospholipid was demonstrated experimentally in quantitative agreement with predicted results. The model is useful in that it rationalizes well the seemingly unusual properties of prothrombinase in straightforward physical terms, provides a means of rationally choosing experimental conditions to both further test and refine the model, and explores the properties not only of prothrombinase but also other blood-clotting or surface-bound enzymatic complexes.
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PMID:"Clotspeed," a mathematical simulation of the functional properties of prothrombinase. 669 15

The coagulation protein Factor Va forms the receptor for the serine protease Factor Xa at the platelet surface. This membrane-bound complex of Factor Va and Factor Xa plus calcium constitutes the enzymatic complex prothrombinase, which effects the conversion of prothrombin to the clotting enzyme, thrombin. Studies were undertaken to investigate the proteolytic events accompanying the inactivation of platelet-bound Factor Va by activated protein C as well as the ability of Factor Xa to protect Factor Va from activated protein C inactivation. During the course of these studies, observations were made which indicated that Factor Va was also cleaved by both a platelet-associated protease, as well as Factor Xa. When Factor Va was incubated with washed platelets, electrophoresis and autoradiography of solubilized platelet pellets indicated that three Factor Va peptides were associated with the platelet: component D (Mr = 94,000), component E (Mr = 74,000), and a 90,000-dalton peptide (component D') which appeared with time as the result of a platelet-associated protease cleavage of component D. The Factor Va peptides bound to platelets were proteolytically inactivated by activated protein C, resulting in five peptide products, all of which remained associated with the platelet-membrane surface. Factor Va was protected from activated protein C proteolysis by complex formation with Factor Xa or active site-blocked Factor Xa. However, active Factor Xa cleaved platelet-bound Factor Va to peptide products which also remained associated with the platelet. Whereas activated protein C rapidly cleaved components D and D' with secondary cleavages occurring in component E, Factor Xa rapidly cleaved component E with secondary cleavages occurring in components D and D'. The Factor Xa-cleaved Factor Va is catalytically functional. To determine whether cleavage was necessary for function, prothrombin conversion reaction mixtures were monitored for thrombin formation and Factor Va cleavage with time in a defined phospholipid vesicle model system. The results indicated that Factor Xa cleavage of Factor Va is not essential for Factor Va activity but may promote its ability to function in the prothrombinase complex.
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PMID:Proteolytic alterations of factor Va bound to platelets. 684 22

We described in a foregoing report findings on serpin, a serine protease inhibitor, newly identified in horseshoe crab (Tachypleus tridentatus) hemocytes and we name it limulus intracellular coagulation inhibitor, LICI (Miura, Y., Kawabata, S., and Iwanaga, S. (1994) J. Biol. Chem. 269, 542-547). This serpin specifically inhibits limulus lipopolysaccharide-sensitive serine protease, factor C. In ongoing studies on limulus serpin, we have found another inhibitor, LICI type-2 (LICI-2), which inhibits not only factor C (k1 = 7.1 x 10(4) M-1 S-1) but also limulus clotting enzyme (k1 = 4.3 x 10(5) M-1 S-1). LICI-2 inhibits mammalian serine proteases, including alpha-thrombin, salivary kallikrein, plasmin, and tissue plasminogen activator. The inactivation of plasmin is the most rapid (k1 = 1.2 x 10(6) M-1 S-1). The purified LICI-2 is a single chain glycoprotein with an apparent M(r) = 42,000. A cDNA for LICI-2 was isolated and the open reading frame coded for a mature protein of 386 amino acids, of which 160 residues were confirmed by peptide sequencing. Although LICI-2 shows significant sequence similarity to the previous limulus serpin, LICI-1 (42% identity), LICI-2 contains a unique putative reactive site, -Lys-Ser-, distinct from that of LICI-1 (-Arg-Ser-). Northern blotting revealed expression of LICI-2 mRNA only in hemocytes, and not in heart, brain, stomach, intestine, coxal gland, and skeletal muscle. The immunoblot of large and small granule components with antiserum against purified LICI-2 suggests that LICI-2 is stored specifically in large granules, as in the case of LICI-1, and is released in response to external stimuli. We propose that the LICIs be classified into a new subfamily of intracellular serpins, regulated secretory serpins.
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PMID:A limulus intracellular coagulation inhibitor type 2. Purification, characterization, cDNA cloning, and tissue localization. 782 80

A Limulus intracellular coagulation inhibitor, designated LICI, was isolated from hemocytes of the Japanese horseshoe crab (Tachypleus tridentatus), using three steps of chromatography, including dextran sulfate-Sepharose CL-6B, Sephacryl S-200, and Mono S. LICI is a single-chain glycoprotein with an apparent M(r) = 48,000 estimated by SDS-polyacrylamide gel electrophoresis. It blocks the amidolytic activities of Limulus lipopolysaccharide-sensitive serine protease, factor C, by forming a covalent 1:1 complex with the protease. The second-order rate constant for inhibition of factor C was 2.5 x 10(6) M-1 s-1 at 37 degrees C. LICI also inhibited human alpha-thrombin, rat salivary kallikrein, bovine plasmin, and trypsin but not Limulus clotting enzyme, Limulus factor B, bovine factor Xa, human factor XIa, human tissue plasminogen activator, human urokinase, chymotrypsin, elastase, and papain. Glycosaminoglycans such as heparin and heparan sulfate had no effect on the inhibitory activity. A cDNA coding for LICI was isolated from a hemocyte cDNA library. The open reading frame of the 1,257-base pair cDNA codes for the mature protein of 394 amino acids, of which 223 residues were confirmed by amino acid sequence analysis. LICI shows significant sequence identities to members of the serpin superfamily, such as human plasminogen activator inhibitor type 2 (40%) and human monocyte/neutrophil elastase inhibitor (39%). LICI contains a putative reactive site, -Arg-Ser-, at the corresponding position present in several inhibitors of the serpin superfamily. The subcellular localization, determined using an anti-LICI polyclonal antibody, indicated that LICI colocates with the Limulus serine protease zymogens in large granules in the hemocyte.
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PMID:A Limulus intracellular coagulation inhibitor with characteristics of the serpin superfamily. Purification, characterization, and cDNA cloning. 827 48

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


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