Detection of phenoloxidase (PO) in hemocytes of the clam Venus antiqua

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Resumen

The prophenoloxidase (proPO) system, particularly studied in Arthropoda, is one of the important mechanisms of non-self recognition in the immunological response of some invertebrates. Under natural conditions, the activation of the system involves the stimulation of serine proteases by the presence of LPS and β-1,3 glycans on the surface of microorganisms. It results in the triggering of proPO, which in turn generates peptides responsible of opsonizing and lytic actions. We report here the preliminary analysis of this type of response in hemocytes of the bivalve mollusc Venus antiqua, both in intact cells and in crude lysates. Our strategy was first to monitor PO activity in whole hemocytes. Second, to confirm its presence in hemocyte lysate supernatant (HLS) by Dot and Western blot analysis using polyclonal antibodies against tyrosinase, the commercially available form of PO. Third, to measure its enzymatic activity in HLS by comparing the oxidation of the substrate L-DOPA in the presence and absence of known activators or inhibitors. In this report, we demonstrate PO activity in granular cell subpopulations. In HLS, we quantitate the specific activity of the enzyme by spectromethomertric methods. Finally, immunological characterization of HLS suggests the existence of two PO-like polypeptides, one of 45 kDa and a second one slightly higher.

Idioma originalInglés
Páginas (desde-hasta)17-22
Número de páginas6
PublicaciónMalacologia
Volumen44
N.º1
EstadoPublicada - 2002
Publicado de forma externa

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