Purification and characterization of cytosolic protein-tyrosine kinase from bovine platelets.

A cytosolic protein-tyrosine kinase has been highly purified from bovine platelets using [Val5]angiotensin II as a substrate. The purification procedure involves sequential column chromatography on phosphocellulose, Sephacryl S-200, poly(L-lysine)-agarose, casein-Sepharose 4B and 2',5'-ADP-Sepharose 4B. Analysis of the most highly purified preparations by SDS/polyacrylamide gel electrophoresis revealed a major silver-stained band of molecular mass 71 kDa. This molecular mass was consistent with results obtained from sucrose density gradient centrifugation, indicating that the enzyme exists as a monomer. The purified kinase, called CPTK 71, efficiently phosphorylated tubulin and p36 (calpactin 1 heavy chain). However, it did not phosphorylate H1 histone. Half-maximal enzyme activity was observed at 2.2 microM ATP, and Mn2+, Co2+ and Mg2+ were effective divalent metal ions for the expression of activity. Insulin, epidermal growth factor, and platelet-derived growth factor had little or no effect on the kinase activity of CPTK 71. CPTK 71 had no immunological cross-reactivity with pp60src. These results suggest that CPTK 71 is a novel type of protein-tyrosine kinases among the enzymes so far reported.