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 μM for substrate phosphorylation and for autophosphorylation. Using casein, we determined a Vmax for substrate phosphorylation by pp60c‐src in the range of 1.9–3.4 nmol · min−1· 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 from 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.
|Original language||English (US)|
|Number of pages||8|
|Journal||European Journal of Biochemistry|
|Publication status||Published - Jun 1990|
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