Phenoloxidases of different sizes are modulated by LPS inoculation into Ciona intestinalis tunic and pharynx

In the present study, to further characterize the pro-phenoloxidase (proPO) and active phenoloxidase (PO) involved in the Ciona intestinalis inflammatory response, tunic and pharynx homogenate supernatants were separated on high pressure liquid chromatography and fractions were assayed for the PO activity before and after LPS inoculation, as well as before and after trypsin treatment which activates proPO. The LPS inoculation per se did not significantly change the basal PO activity of the tunic homogenate supernatant (THS) and pharynx homogenate supernatant (PHS) restricted in two confluent peaks, whereas a significant enhancement was observable after the trypsin treatment. This trypsin effect suggests that proPO is the main component of the HPLC separated fractions, and indicates that LPS inoculation mainly challenges the pro-enzyme production by tunic cells and hemocytes, as well as the activation of the serine-protease pathway. The protein size analysis and DOPA-MBTH assay, disclose two active proteins of 90.0 and 170.0 kDa differently contained in the two main chromatographic peaks. Due to the SDS activating effect on the proenzyme analyzed by SDS-PAGE, the size of proPO could not be shown, whereas modulation of an oligomerization process of the 90 kDa component is suggested.