Purification of a tyrosine-specific protein kinase from Rous sarcoma virus-induced rat tumor.

摘要: We have identified a tyrosine kinase activity present in tumors which were raised rats by subcutaneous injection of Rous sarcoma virus-transformed rat cells (SR-NRK). This phosphorylates on the heavy chain IgG from tumor-bearing rabbit (TBR) sera specific for src gene product, pp60src. Using TBR-IgG phosphorylation as an assay, we purified this over 7200-fold. The purification procedure involves detergent extraction followed sequential column chromatography hydroxylapatite, DEAE-Sephacel, oligodeoxyadenosine-cellulose, affinity prepared TBR-sera, and Sephacryl S-200. behaves molecule apparent Mr = 54,000 S-200 molecular sieve chromatography. Analysis fractions SDS-PAGE indicates that major Coomassie blue-stained band with (p54), co-elutes peak activity. From 600 g tumors, approximately 200 micrograms p54 are obtained. four types evidence show is related to 1) Purified capable undergoing endogenous presence [gamma-32P]ATP producing 32P-labeled pp54 polypeptide specifically immunoprecipitated TBR-sera contains only phosphotyrosine. 2) competes pp60src binding TBR-IgG, indicating degree starting material agrees very well results obtained assay. 3) V8 protease digestion suggests they share common 26,000 fragment. 4) Antibodies partially precipitate chicken cells.