Cytotoxicity Of Okadaic Acid And Kinetic Characterization Of Protein Tyrosine Phosphatase Activity In V79 Fibroblasts

Protein phosphorylation and dephosphorylation mediate signal transduction events that control several important cellular processes. The aim of this work was to determine some kinetic properties of a protein phosphatase obtained from non-metabolizing V79 Chinese hamster cells. The effect of okadaic acid, a tumour promoter, on these cell cultures was also determined. Phosphatase activity was assayed in V79 cells which were lysed with 0.1 M imidazole buffer (pH 7.4). Enzyme activity was determined using p-nitrophenylphosphate and tyrosine phosphate (TyrP) as substrates. Maximum phosphatase activity was obtained at pH7·4. The apparent K(m) (Michael's constant) and specificity constant for TyrP were 0·06 nm and 57, respectively. Phosphatase activity was inhibited by 100μM pCMB (p-chloro-mercuribenzoate; 70%), 10 mM fluoride (30%), 10 mM phosphate (40%), 100 μM m-vanadate (80%) and 100 μM o-vanadate (90%). Tartrate (5 mM) had no effect. 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