Vmax. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A.
A kinetic analysis of the tyrosine-specific protein kinase of pp60c-src from the C1300 mouse neuroblastoma cell line Neuro-2A and pp60c-src expressed in fibroblasts was carried out to determine the nature of the increased specific activity of the neuroblastoma enzyme. In immune-complex kinase assays with ATP-Mn2+ and the tyrosine-containing peptide angiotensin I as phosphoacceptor substrate, pp60c-src from the neuroblastoma cell line was characterized by a maximum velocity (Vmax.) that was 7-15-fold greater than the Vmax. of pp60c-src from fibroblasts. The neuroblastoma enzyme exhibited Km values for ATP (16 +/- 3 microM) and angiotensin I (6.8 +/- 2.6 mM) that were similar to Km values for ATP (25 +/- 3 microM) and angiotensin I (6.5 +/- 1.7 mM) of pp60c-src from fibroblasts. pp60v-src expressed in Rous-sarcoma-virus-transformed cells exhibited an ATP Km value (25 +/- 4 microM) and an angiotensin I Km value (6.6 +/- 0.5 mM) that approximated the values determined for pp60c-src in neuroblastoma cells and fibroblasts. These results indicate that the pp60c-src kinase from neuroblastoma cells has a higher turnover number than pp60c-src kinase from fibroblasts, and that the neural form of the enzyme would be expected to exhibit increased catalytic activity at the saturating concentrations of ATP that are found intracellularly.
Matten
W T
WT
Department of Biochemistry, University of North Carolina School of Medicine, Chapel Hill 27514.
Maness
P F
PF
D000255:Adenosine Triphosphate / Q000493:pharmacokinetics; D000803:Angiotensin I / Q000493:pharmacokinetics; D000818:Animals; D002460:Cell Line; D004789:Enzyme Activation; D005347:Fibroblasts / Q000201:enzymology; D007122:Immunoelectrophoresis; D007700:Kinetics; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D011518:Proto-Oncogene Proteins; D000255:Adenosine Triphosphate; D000803:Angiotensin I; D011505:Protein-Tyrosine Kinases