title: Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes.
abstract: 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.
mesh_terms: D000596:Amino Acids / Q000032:analysis; D000818:Animals; D060829:Arthropod Proteins; D001777:Blood Coagulation / Q000187:drug effects; D001779:Blood Coagulation Factors / Q000032:analysis / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification*; D004792:Enzyme Precursors / Q000032:analysis / Q000302:isolation & purification; D006434:Hemocytes / Q000032:analysis; D006737:Horseshoe Crabs / Q000032:analysis*; D007531:Isoflurophate / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology; D008970:Molecular Weight; D012697:Serine Endopeptidases; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D060829:Arthropod Proteins; D001779:Blood Coagulation Factors; D004792:Enzyme Precursors; D008070:Lipopolysaccharides; D007531:Isoflurophate; D010450:Endopeptidases; D012697:Serine Endopeptidases; C036776:Limulus clotting factor B