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