Characterization of purified pp60c‐src protein tyrosine kinase from human platelets

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Abstract
Intact pp60c-src, the cellular homologue of the transforming protein of Rous sarcoma virus, was purified from human platelets. The purified fractions also contained small amounts of a 54-kDa proteolytic degradation product of pp60c-src. We investigated some of the biochemical and kinetic properties of pp60c-src protein tyrosine kinase. Maximum kinase activity occurred at pH 6.5 and required a mixture of 2 mM Mn2+/Mg2+ as divalent cations. The enzyme most strongly phosphorylated casein, followed by enolase and alcohol dehydrogenase. The Km value for ATP was 4 .mu.M for substrate phosphorylation and for autophosphorylation. Using casein, we determined a Vmax for substrate phosphoorylation by pp60c-5rc in the range of 1.9-3.4 nmol .cntdot. min-1 .cntdot. mg -1. Since the Vmax value for the purified 54-kDa fragment of pp60c-src was also included in this value, we conclude that proteolytic degradation of a 6-kDa fragment form the N-terminus of pp60c-src did not affect its kinase activity. Tryptic phosphopeptide analysis identified Tyr-416 as the major autophosphorylation site. Preincubation of purified pp60c-src with ATP increased the amount of autophosphorylation accompanied by an increase in Vmax, whereas the Km values were not altered. Our data directly demonstrate that autophosphorylation at Tyr-416 exerts, in contrast to phosphorylation at Tyr-527, a positive regulatory effect on the pp60c-src kinase activity.