Partial purification and characterization of pro-phospholipase A2 activating proteases from gill membranes of the red sea bream, Chrysophrys major

We previously reported that gill group IB secretory phospholipase A2 (sPLA2) exists as an inactive pro-sPLA2 with the dipeptide Ala-Arg, at the N-terminus of mature sPLA2 in mucous cells. Pro-sPLA2 should be activated after being secreted to the surface of gill epithelia by trypsin-like protease. To clarify the above hypothesis, we investigated the existence of pro-sPLA2 activating protease (PAP) in the gills of the red sea bream, using gill pro-sPLA2 as a substrate. PAP was solubilized from the membrane fraction of the gills with 2% sodium cholate and partially purified by benzamidine-Sepharose chromatography and reversed-phase HPLC. Partially purified proteases, PAP1 and PAP2 showed a high molecular mass of about 200 kDa by gelatin zymography. PAP1 and PAP2 had optimal pH from 7 to 9 and were inhibited by trypsin inhibitors. These properties of PAP1 and PAP2 suggest that both enzymes belong to the membrane-associated trypsin-like serine protease family, such as enteropeptidase and corin. This is the first report verifying the existence of the activating protease of group IB pro-sPLA2 isoforms in a non-digestive tissue.