Characterization of purified pp60c-src protein tyrosine kinase from human platelets.
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 microM 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.
Rudolf Buchheim-Institut für Pharmakologie, Justus-Liebig-Universität, Giessen, Federal Republic of Germany.
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D000255:Adenosine Triphosphate / Q000494:pharmacology; D001665:Binding Sites; D001792:Blood Platelets / Q000032:analysis*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000032:analysis; D010449:Peptide Mapping; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification*; D011518:Proto-Oncogene Proteins / Q000302:isolation & purification*; D016392:Proto-Oncogene Proteins pp60(c-src); D014357:Trypsin; D014443:Tyrosine / Q000378:metabolism
D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D014443:Tyrosine; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D016392:Proto-Oncogene Proteins pp60(c-src); D014357:Trypsin