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)

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

An intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr = 64,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr = 64,000, 40,000, and 25,000) were detected on SDS-PAGE after reduction. This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr of 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64,000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.
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PMID:Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes. 351 94

A proclotting enzyme associated with the hemolymph coagulation system of limulus (Tachypleus tridentatus) was highly purified from the hemocyte lysate. The first step of purification was performed by chromatography of the lysate on a pyrogen-free dextran sulfate-Sepharose CL-6B column, which was essential for separation of the proclotting enzyme from its activator, named factor B. The following steps consisted of column chromatographies on DEAE-Sepharose CL-6B, Sephadex G-150, benzamidine-CH-Sepharose and Sephacryl S-300. Through these procedures, 1.4 mg of the purified material was obtained from 630 ml of the lysate and approximately 300-fold purification was achieved. The preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence and absence of 2-mercaptoethanol. The single-chain proclotting enzyme was a glycoprotein with an apparent molecular weight of 54,000, and no gamma-carboxyglutamic acid was detected. The proclotting enzyme was converted to its active form by purified factor B or by trypsin. The resulting clotting enzyme had a molecular weight of 54,000, consisting of a heavy chain of Mr = 31,000 and a light chain of Mr = 25,000. The serine active site of the clotting enzyme was found in the heavy chain. The chemical analyses of the isolated heavy and light chains indicated that the activation of the proclotting enzyme to its active form by factor B or trypsin is induced by a limited proteolysis, yielding two chains bridged by a disulfide linkage(s).
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PMID:Intracellular proclotting enzyme in limulus (Tachypleus tridentatus) hemocytes: its purification and properties. 403 Jul 38