The prophenoloxidase system is activated during the tunic inflammatory reaction of Ciona intestinalis

Phenoloxidase (PO) activity was examined in thetunic tissue of Ciona intestinalis following lipopolysaccharide(CinPO-3) containing the CinPO-2 peptide was identifiedin the recent Ciona genome version. Presumably, LPSstimulated the production and dimerization (120 kDa) ofCinPO-3 (66 kDa). Thus, the activated proPO systemincludes several POs that are distinguishable by size andthat are contained and presumably released by tunicinflammatory cells and hemocytes of the pharynx bars.(LPS) intratunic injection. Tunic homogenate supernatant(THS), assayed with the Dopa-MBTH reaction,displayed Ca2+-independent PO activity that was raised byLPS and further enhanced by proteases. Specific inhibitors(tropolone, phenylthiourea, diethylthiocarbamate) supportedthe specificity of the reaction. Assay with soybeantrypsin inhibitor showed that, in the tunic, PO activationwith trypsin was not significantly inhibited suggesting thatproteases diverse from serine proteases were involved. Invivo experiments were carried out by injecting isosmoticmedium or LPS, and THS was assayed for its PO activity.Analysis of variance of the time-course profiles showed thatLPS was more effective in activating proPO. To disclosethe PO response at the injured site, an assay with Dopa-MBTH was performed in vitro. Quinones were mainlycontained in the tunic matrix enriched with inflammatorycells around the injection site. Microscopic observationsand immunohistochemistry with anti-CinPO-2 antibodiesshowed granulocytes and unilocular refractile granulocytescontaining PO, whereas few morula cells were stained. InTHS zymograms (SDS-polyacrylamide gel electrophoresis),PO activity linked to 90-kDa and 120-kDa bands wasobserved as an effect of LPS injection, whereas the densityof 170-kDa PO was weak. A third presumptive PO enzyme