Fractionation of Limulus amebocyte lysate. Characterization of activation of the proclotting enzyme by an endotoxin-mediated activator

Abstract

Limulus amebocyte lysate was fractionated by heparin-Sepharose chromatography into four components (fractions A, B, C and D). Major coagulation factors, i.e., proclotting enzyme, coagulogen, and proclotting enzyme activating factor precursor (proactivator) in the lysate were eluted, respectively, in fraction A, fraction B and fraction C. Clotting enzyme activity was detected only following recombination of fraction A and fraction C in the presence of endotoxin. The conversion of proactivator to its active form (activator) was an endotoxin-dependent reaction and was inhibited by polymyxin B. Either proactivator is an endotoxin-sensitive factor or another endotoxin-sensitive factor, which activates proactivator, is present in fraction C. Optimal pH for proclotting enzyme activation by activator was broad and ranged from pH 6.0 to 8.0, while that for the endotoxin-mediated activation of proactivator was pH 7.0. No initial latent period was observed during activation of the proactivator or proenzyme. The activator was inhibited by benzamidine, leupeptin, soybean trypsin inhibitor and diisopropyl fluorophosphate, suggesting that the activator is a trypsin-type serine protease. Trypsin, but not thrombin, urokinase, plasmin, papain or alpha-chymotrypsin activated the proclotting enzyme. Therefore, limited proteolysis, i.e., of an arginyl- or lysyl-X bond(s), of the proenzyme molecule is probably involved in its activation.