Differential tissue distribution and specificity of phenoloxidases from the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) play a key role in melanin production, are involved in invertebrate immune mechanisms, and have been detected in different bivalves. Recently, we identified catecholase- and laccase-like PO activities in plasma and haemocyte lysate supernatant (HLS) of the Pacific oyster Crassostrea gigas. logo further in our investigations, the aims of this study were (i) to determine the tissue distribution of PO activities in C gigas, and (ii) to identify and characterise the different sub-classes of POs (i.e. tyrosinase. catecholase and/or laccase) involved in these oxido-reductase activities. With dopamine and p-phenylenediamine (PPD) but not with L-tyrosine used as substrates, PO-activities were detected by spectrophotometry in the gills, digestive gland, mantle, and muscle. These results suggest the presence of catecholase and laccase but not of tyrosinase activities in oyster tissues. The highest activity was recovered in the digestive gland. PO-like activities were all inhibited by 1-phenyl-2-thiourea (PTU) and by the specific laccase inhibitor, cethyltrimethylammonium bromide (CAB). With dopamine as substrate, the catecholase inhibitor 4-hexylresorcinol (4-HR) only inhibited PO in the muscle. SDS-PAGE zymographic assays with dopamine and PPD elicited a unique similar to 40 kDa protein band in the muscle. In the other tissues, laccase-like activities could be related to similar to 10 kDa and/or similar to 200 kDa protein bands. The similar to 10 kDa protein band was also detected in plasma and HLS, confirming the presence of a laccase in the later compartments, and probably in most of the tissues of C gigas. This is the first time to our knowledge that a similar to 10 kDa protein band is associated to a laccase-like activity in a mollusc species, contributing to the characterisation of phenoloxidase activities in marine bivalves. (C) 2011 Elsevier Inc. All rights reserved.