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 lipopolysaccharide (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 zymosan. Phenoloxidase activity was blocked by serine protease inhibitors, such as soybean trypsin inhibitor (STI), leupeptin, and phenylmethyl-sulphonylfluoride (PMSF), and substantially increased by cysteine protease inhibitors (N-methylmaleimide, N-ethylmaleimide, and iodoacetamide). This enhancement was observed only when the proPO system was activated. Incubation without activators or preincubation with STI prevented the induced enhancement. Electrophoretic analyses of HLS treated with zymosan or LPS showed that three bands at 41, 39, and 37 kDa were specifically modified when the system was activated. These results suggest that a serine protease is involved in the activation of the proPO system in P. clarki, and a mechanism susceptible to cysteine protease inhibitors could be related to its regulation.