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 A(2) (sPLA(2)) exists as an inactive pro-sPLA(2) with the dipeptide Ala-Arg, at the N-terminus of mature sPLA(2) in mucous cells. Pro-sPLA(2) 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-sPLA(2) activating protease (PAP) in the gills of the red sea bream, using gill pro-sPLA(2) 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-sPLA(2) isoforms in a non-digestive tissue.
Uchiyama
Satoshi
S
Laboratory of Molecular Cell Biology, Graduate School of Biosphere Science, Hiroshima University, 1-4-4 Kagamiyama, Higashihiroshima 739-8528, Japan.
Iijima
Noriaki
N
D000818:Animals; D004789:Enzyme Activation; D005880:Gills / Q000201:enzymology*; D007527:Isoenzymes / Q000378:metabolism; D000073718:Pancreatitis-Associated Proteins; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010741:Phospholipases A / Q000737:chemistry / Q000378:metabolism*; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism; D021541:Sea Bream; D013552:Swine; D014357:Trypsin / Q000378:metabolism
D007527:Isoenzymes; D000073718:Pancreatitis-Associated Proteins; D011498:Protein Precursors; C000612141:REG3A protein, human; D010741:Phospholipases A; D010447:Peptide Hydrolases; D014357:Trypsin