Inter- and intramolecular interactions of highly purified Rous sarcoma virus-transforming protein, pp60v-src.
We have purified intact pp60v-src, the product of the Rous sarcoma virus src gene, over 2400-fold, based on the phosphorylation of tumor-bearing rabbit IgG. The purification procedure involved detergent extraction of the particulate fraction of the cells and sequential chromatography on hydroxylapatite, butyl agarose, DEAE-Sephacel, ADP-agarose, and Sephacryl S-200. Analysis of the preparation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single silver-stained band with an apparent molecular weight of 60,000. Our results show that the activities of this preparation were qualitatively similar to those described previously for partially purified pp60v-src. Upon analysis by two-dimensional gel electrophoresis, the purified pp60v-src yielded one major species which migrated to the same position as the least acidic of the three major species detectable in cellular lysates, suggesting that the pp60v-src had been dephosphorylated during the purification procedure. We found that pp60v-src was very prone to aggregation; to maintain it as a monomer both Nonidet P-40 and KCl were required. Under conditions which maintained pp60v-src as a monomer, the rate of autophosphorylation was independent of its concentration and thus proceeded via an intramolecular process. Preincubation of pp60v-src with ATP or GTP as well as nonphosphorylating analogs of ATP or GTP preserved its phosphorylating activity toward alpha-casein whereas its activity was reduced 80% upon preincubation in the absence of nucleotides. We suggest that protection with nucleotides rather than autophosphorylation accounts for the apparent increase in the activity of pp60v-src after incubation of the enzyme with ATP.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D003411:Arvicolinae; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000494:pharmacology; D015688:Oncogene Protein pp60(v-src); D010766:Phosphorylation; D011817:Rabbits; D012191:Retroviridae Proteins / Q000032:analysis* / Q000302:isolation & purification
D012191:Retroviridae Proteins; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D015688:Oncogene Protein pp60(v-src)