PROPHENOLOXIDASE SYSTEM ACTIVATION IN THE CRAYFISH PROCAMBARUS-CLARKI

The prophenoloxidase system (proPO) was studied in primary cultures of hemocytes of the crayfish Procambarus clarki. Both zymosan and lipopo lysaccharide (LPS) separately induced rapid degranulation and lysis of semigranular hemocytes, with concurrent release of proPO. ProPO could be demonstrated in the hemocyte lysate supernatant (HLS) obtained by a freeze/thaw method, and was specifically activated by LPS and zymosa n. Phenoloxidase activity was blocked by serine protease inhibitors, s uch as soybean trypsin inhibitor (STI), leupeptin, and phenylmethyl-su lphonylfluoride (PMSF), and substantially increased by cystein proteas e inhibitors (N-methylmaleimide, N-ethylmaleimide, and iodoacetamide). This enhancement was observed only when the proPO system was activate d. Incubation without activators or preincubation with STI prevented t he induced enhancement. Electrophoretic analyses of HLS treated with z ymosan or LPS showed that three bands at 41, 39, and 37 kDa were speci fically modified when the system was activated. These results suggest that a serine protease is involved in the activation of the proPO syst em in P. clarki, and a mechanism susceptible to cystein protease inhib itors could be related to its regulation.