Abstract
We describe here the conditions of thin layer chromatography (TLC) and high pressure liquid chromatography (HPLC) to improve the analytical method of phosphotyrosine (P-Tyr) in biological sample. TLC was performed on silica plate with the mixture of propanol and water (2.1∶1 v/v) as a mobile phase and R1 values were 0.42, 0.39 and 0.33 for phosphotyrosine, phosphothreonine and phosphoserine, respectively. HPLC was performed on NH2 column with a mobile phase of potassium biphosphate solution by UV detection at 192 nm. The optimum condition of HPLC was obtained at 0.01 M, pH 4.5 with a clear separation within 12 min. These procedures have been applied to the analysis of phosphotyrosine obtained from tyrosine-phosphorylated enolase. Both TLC and HPLC methods were suitable to analyze tyrosine-phosphorylated protein without being affected by contaminants from hydrolysates.
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References Cited
Blithe, D. L., Richard, N. D., and Pastan, I. H., Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor.J. Biol. Chem., 257, 7135–7142 (1982).
Hunter, T., and Sefton, B. M., Transforming gene product of Rous sarcoma virus phosphorylates tyrosine.Proc. Natl. Acad. Sci. USA, 77, 1311–1315 (1980).
Imes, S. S., Kathan, N. O., and Knowles, A. F., Plasma membrane-associated phosphatase activities hydrolyzing [32P] phosphatidyl histones and [32P] phosphatidylinositol phosphate.Anal. Biochem., 161, 316–322 (1987).
Jacobs, S., Kull, Jr., F. C., Eaarp, H. S., Svoboda, M. E., Van Wyk, J. J., and Custrecasas, P., Somatomedin-C stimulates the phosphorylation of the β-subunit of its own receptor.J. Biol. Chem., 258, 9581–9584 (1983).
Kasuga, M., Yamaguchi, Y. F., Blithe, D. L., White, M. F., and Kahn, C. R., Characterization of the insulin receptor kinase purified from human placenta membranes.J. Biol. Chem., 258, 10973–10980 (1983).
Kasuga, M., Zick, Y., Blithe, D. L., Karlesson, F. A., Haring, H. U., and Kahn, C. R., Insulin stimulation of phosphorylation of the β subunit of the insulin receptor: Formation of both phosphoserine and phosphotyrosine.J. Biol. Chem., 257, 9891–9894 (1982).
Martensen, T. M., Phosphotyrosine in proteins: Stability and quantification.J. Biol. Chem., 257, 9648–9652 (1982).
Mitchell, H. K., and Lunan, K. D., Tyrosine-o-phosphate in Drosophila.Arch. Biochem. Biophys., 106, 219–222 (1964).
Munoz, G., and Marshall, S. H., An alternative method for a fast separation of phosphotyrosine.Anal. Biochem., 190, 233–237 (1990).
Richert, N. D., Blithe, D. L., and Pastan, I., Properties of the src kinase purified from Rous sarcoma virus-induced rat tumors.J. Biol. Chem., 257, 7143–7150 (1982).
Robert, J. C., Soumarmon, A., Lewin, M. J. M., Determination ofo-phosphothreonine,o-phosphoserine,o-phosphotyrosine and phosphate by high performance liquid chromatography.J. Chromatogr., 338, 315–324 (1985).
Rothberg, P. G., Harris, T. J. R., Nomoto, A., and Wimmer, E.O 4-(5′-uridyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus.Proc. Natl. Acad. Sci. USA, 75, 4868–4872 (1978).
Sasak, N., Ress-Jones, R. W., Zick, Y., Nissley, S. P., and Rechler, M. M., Characterization of insulin-like growth factor 1-stimulated tyrosine kinase activity associated with the β-subunit of type 1 insulin-like growth factor receptors of rat liver cells.J. Biol. Chem., 260, 9793–9804 (1985).
Swarup, G., Cohen, S., and Garbers, D. L., Selective dephosphorylation of protein containing phosphotyrosine by alkaline phosphatases.J. Biol. Chem., 256, 8197–8201 (1981).
Swarup, G., Dasgupta, J. D., and Garbers, D., Tyrosine protein kinase activity of rat spleen and other tissuses.J. Biol. Chem., 258, 10341–10347 (1983).
Tonks, N. K., Diltz, C. D., and Fisher, E. H., Purification of the major protein-tyrosine-phosphatases of human placenta.J. Biol. Chem., 263, 6722–6730 (1988).
Ushiro, H., and Cohen, S., Identification of phosphotyrosine as a product of epidermal growth factor-activated protein kinase in A-431 cell membranes.J. Biol. Chem., 255, 8363–8365 (1980).
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Song, YM., Yoo, GS., Lee, SK. et al. Improved phosphotyrosine analysis by TLC and HPLC. Arch. Pharm. Res. 16, 99–103 (1993). https://doi.org/10.1007/BF03036854
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DOI: https://doi.org/10.1007/BF03036854