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Pia EKMAN, Bo EK, Lorentz ENGSTRÖM, The Quantity of Protein-Bound [ 32 P]Phosphotyrosine in Hepatocytes and Fibroblasts. The Effects of Tyrosine Protein Kinase Activating Agents , The Journal of Biochemistry, Volume 101, Issue 4, 1987, Pages 863–870, https://doi.org/10.1093/oxfordjournals.jbchem.a121953
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Abstract
Tyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32 P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectro-photometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [ 32 P]phosphotyrosine could be calculated. The method proved to be useful for the determination of [ 32 P]phosphotyrosine is small amounts of cells. The minimum detectable amount of [ 32 P]phosphotyrosine was about 1 pmol, and as an example, only 2.5 × 10 6 fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [ 32 P]phos-photyrosine from 600 to 3,200 pmol/g of fibroblasts, while insulin only increased the [ 32 P]phosphotyrosine from 110 to 120 pmol/g of hepatocytes.
