Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin.
An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.
Tokunaga
F
F
Department of Biology, Faculty of Science, Kyushu University, Fukuoka.
Nakajima
H
H
Iwanaga
S
S
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D060829:Arthropod Proteins; D002918:Chymotrypsin / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004792:Enzyme Precursors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006434:Hemocytes / Q000201:enzymology; D006737:Horseshoe Crabs / Q000201:enzymology; D007700:Kinetics; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D013379:Substrate Specificity; D014357:Trypsin / Q000494:pharmacology
D000596:Amino Acids; D060829:Arthropod Proteins; D004792:Enzyme Precursors; D008070:Lipopolysaccharides; D012697:Serine Endopeptidases; D002918:Chymotrypsin; D014357:Trypsin; C057151:Limulus clotting factor C