PROTEOLYTIC-ENZYMES IN THE BLOOD-FEEDING PARASITIC COPEPOD, PHRIXOCEPHALUS-CINCINNATUS

To understand digestive and invasive mechanisms employed by blood-feed ing parasitic copepods, extracts of Phrixocephalus cincinnatus were as sayed for specific proteolytic enzyme activity. Intact parasites were dissected into the 3 major body regions, cephalothorax (CT), genital s egment (GS), and eggstrings (ES), and homogenized in ice-cold 1% Trito n X-100 (v/v) in water Protease activity in each body region was assay ed using several synthetic fluorogenic peptide substrates. The greates t activity was detected when samples were incubated with carbobenzoxy phenylalanyl-arginyl-7-amido-4-methylcoumarin (CBZ-phe-arg-NHMec) in t he presence of cysteine or reducing agents. Substrate specificity, pH profile, and inhibitor sensitivity indicated that the proteolytic enzy me(s) belonged to the cysteine class of endopeptidases and were most s imilar to mammalian cathepsins L, B, and H, respectively. intense prot ease activity was also detected with -glycyl-L-prolyl-L-arginine-7-ami do-methylcoumarin (CBZ-gly-pro-arg-NHMec), a substrate for the serine proteases, plasmin and thrombin. Substrate gel electrophoresis reveale d intense gelatinolytic activity in all body regions; however, the ES extract presented a pattern different from that of the adult body, sug gesting that distinct proteolytic enzymes are expressed during develop ment Gelatinolytic activity was inhibited at low pH and in the presenc e of serine protease inhibitors but not cysteine protease inhibitors. Collectively, the results indicate the presence of 2 major classes of proteolytic enzymes, cysteine and serine proteases. Differential expre ssion of these proteases may be important for the successful completio n of the parasite's lift cycle, as well as survival of the adult.