Open AccessLipopolysaccharide‐sensitive serine‐protease zymogen (factor C) found in Limulus hemocytes
Author(s) -
NAKAMURA Takanori,
MORITA Takashi,
IWANAGA Sadaaki
Publication year - 1986
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1986.tb09427.x
Subject(s) - limulus , zymogen , hemolymph , serine protease , complement factor b , protease , biochemistry , lipopolysaccharide , sodium dodecyl sulfate , prophenoloxidase , polyacrylamide gel electrophoresis , factor x , microbiology and biotechnology , chemistry , gel electrophoresis , thrombin , virulence factor , biology , enzyme , complement system , platelet , immunology , innate immune system , antibody , paleontology , receptor , virulence , gene
Bacterial endotoxin (lipopolysaccharide, LPS) induces coagulation of horseshoe crab hemolymph. Our previous studies had demonstrated that a hemolymph factor, designated factor B, was associated with the LPS‐mediated activation of the Limulus clotting system [Ohki et al. (1980) FEBS Lett. 120 , 318–321]. On further purification of factor B we found that an additional component, designated factor C, was required to generate factor B activity in the presence of LPS in order to activate the proclotting enzyme. To elucidate the role of factor C in the LPS‐mediated reaction, factor C was isolated and characterized from the hemocyte lysate under sterile conditions. The preparation exhibited a single band on sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS‐PAGE) in the absence of 2‐mercaptoethanol, while two protein bands on SDS‐PAGE were observed after reduction. Thus, factor C had a M r of 123000 consisting of a heavy chain of M r = 80000 and a light chain of M r = 43000. Factor C was converted to an activated form in the presence of LPS with a M r = 123000, designated factor C̄. Upon activation, cleavage of the light chain occurred resulting in the accumulation of two new fragments of M r = 34000 and 8500 on reduced SDS‐PAGE. A diisopropylfluorophosphate‐sensitive active site was localized in the light chain ( M r = 34000) of factor C̄. The reconstitution experiments, using factor C, factor B, proclotting enzyme and LPS, demonstrated that all of these proteins are essential for the endotoxin‐mediated coagulation system. On the basis of these results we propose that a cascade pathway of LPS‐induced activation of the Limulus clotting system consists of three sequential activations of hemolymph serine protease zymogens.