Purification of Limulus polyphemus proclotting enzyme.
Horseshoe crabs (Limulus polyphemus and Tachypleus tridentatus) possess a proteolytic blood coagulation system within their amebocytes that, after release and endotoxin activation, generates a polymerized insoluble coagulin clot. Clotting enzyme from horseshoe crab amebocyte lysate is the protease that activates the clottable protein (coagulogen) which then forms the coagulin clot. Comparison of the previously published descriptions of this enzyme has revealed significantly discordant biochemical characteristics. We purified a 60-kDa proclotting enzyme from L. polyphemus amebocyte lysate to a single band on polyacrylamide gel electrophoresis. After electrophoresis, evaluation of enzymatic activity of this protein within gels demonstrated that the band of purified protein corresponded to enzymatic activity, as detected by amidolytic activity for chromogenic substrates and by gelation of coagulogen applied to the gel. The enzymatic activity was inhibited by serine protease inhibitors. The purified proclotting enzyme had a molecular weight and amino acid composition different from the previously published characterizations of proclotting enzymes from both L. polyphemus and T. tridentatus.
Roth
R I
RI
Department of Laboratory Medicine, University of California School of Medicine, San Francisco.
Levin
J
J
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D004792:Enzyme Precursors / Q000302:isolation & purification* / Q000378:metabolism; D006737:Horseshoe Crabs / Q000201:enzymology*; D007700:Kinetics; D008033:Limulus Test; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D014357:Trypsin / Q000378:metabolism
D000596:Amino Acids; D004792:Enzyme Precursors; D011480:Protease Inhibitors; D010450:Endopeptidases; D014357:Trypsin; C014775:pro-clotting enzyme