Protein kinase C in the spleen of the turbot (Scophthalmus maximus L.)

• Published in: Fish physiology and biochemistry Vol. 20; no. 2; pp. 101 - 114
• Main Authors: Puente-novoa, Jm, Barja, P
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Nature B.V 01.02.1999
• Subjects: Cellulose; Kinases; Marine; Proteins; Scophthalmus maximus; Spleen
• ISSN: 0920-1742; 1573-5168
• DOI: 10.1023/A:1007759610249

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 μg ml-1) and either DAG (2 μg ml-1) or PMA (2 μg 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 (α, β, δ, ε, [theta], μ, ζ and λ) indicated the presence of all these isozymes in turbot spleen.[PUBLICATION ABSTRACT]