Foulkes, J. Gordon and Chow, Marie and Gorka, Carolyn and Frackelton, A. Raymond, Jr. and Baltimore, David (1985) Purification and characterization of a protein-tyrosine kinase encoded by the Abelson murine leukemia virus. Journal of Biological Chemistry, 260 (13). pp. 8070-8077. ISSN 0021-9258. http://resolver.caltech.edu/CaltechAUTHORS:FOUjbc85
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Sequences termed v-abl, which encode the protein-tyrosine kinase activity of Abelson murine leukemia virus, have been expressed in Escherichia coli as a fusion product (ptabl50 kinase). This fusion protein contains 80 amino acids of SV40 small t and the 403 amino acid protein kinase domain of v-abl. We report here the purification and characterization of this kinase. The purified material contains two proteins (Mr = 59,800 and 57,200), both of which possess sequences derived from v-abl. Overall purification was 3,750-fold, with a 31% yield, such that 117 micrograms of kinase could be obtained from 40 g of E. coli within 6-7 days. The specific kinase activity is over 170 mumol of phosphate min-1 mumol-1, comparable to the most active protein- serine kinases. Kinase activity is insensitive to K+, Na+, Ca2+, Ca2+- calmodulin, cAMP, or cAMP-dependent protein kinase inhibitor. The Km for ATP is dependent on the concentration of the second substrate. GTP can also be used as a phosphate donor. The enzyme can phosphorylate peptides consisting of as few as two amino acids and, at a very low rate, free tyrosine. Incubation of the kinase with [gamma-32P]ATP results in incorporation of 1.0 mol of phosphate/mol of protein. This reaction, however, cannot be blocked by prior incubation with unlabeled ATP. Incubation of 32P-labeled kinase with either ADP or ATP results in the synthesis of [32P]ATP. This suggests the phosphotyrosine residue on the Abelson kinase contains a high energy phosphate bond.
|Additional Information:||Copyright © 1985 by the American Society for Biochemistry and Molecular Biology. (Received for publication, November 13, 1984) We would like to thank Sy-dar Wang and Daniel Wang (Massachusetts Institute of Technology, Cambridge, MA) for use of their fermentor and their help in growing the bacteria used in these studies. We would also like to acknowledge Jean Wang (University of California, San Diego, CA) for providing the E. coli containing the ptabl50 kinase plasmid, and Tom Soderling for phosphorylase kinase and Jackie Corbin (Howard Hughes Institute, Nashville, TN) for the catalytic subunit of the CAMP-dependent protein kinase. Finally, we would like to thank John Port (Massachusetts Institute of Technology, Cambridge, MA) for his help in the analysis of the enzyme kinetic data. This work was supported by Program Project Grant CA 26717 from the National Cancer Institute. Part of this work was carried out while J.G.F. was a recipient of a Medical Research Council (United Kingdom) Travel Fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.|
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|Deposited On:||13 Aug 2008 04:22|
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