Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.) |
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Authors: | JM Puente-Novoa P Barja |
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Institution: | (1) Departamento de Bioquímica y Biología Molecular, Facultad de Biología, Universidad de Santiago de Compostela, Campus Sur. 15706 Santiago de Compostela, Galicia, Spain |
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Abstract: | PKC activity was detected in spleen extracts from the turbot, Scophthalmus maximus, a teleost flatfish that is farmed commercially in several countries, in assays with the substrate EGF- R651–658 as phosphate acceptor. The activity was purified about 700-fold by a three-step chromatographic procedure (DEAE-cellulose, phenyl-Sepharose and threonine-Sepharose). Maximal activity was obtained in the presence of the typical PKC cofactors Ca2+ (0.1 mM) PtdS (20 g ml–1) and either DAG (2 g ml–1) or PMA (2 g ml–1). Activity was dose-dependently inhibited by H7 and by the PKC-specific inhibitors PKC19–36 and N-myristoylated PKC19–31. The rate of phosphorylation was highest with the PKC-specific substrate MARCKS161–175. In immunoblotting, MC5 (a mouse monoclonal antibody raised against bovine PKC) recognized bands of 80 and 100 kDa. Immunoblotting with antibodies raised against mouse PKC isozymes (, , , , , , and ) indicated the presence of all these isozymes in turbot spleen. |
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Keywords: | cellular regulation immune tissues protein kinase C isoenzymes protein purification signal transduction teleost biochemistry |
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