Inter- and intramolecular interactions of highly purified Rous sarcoma virus-transforming protein, pp60v-src

Area of Science:

• Molecular Biology
• Virology
• Biochemistry

Background:

• pp60v-src is the protein product of the Rous sarcoma virus src gene.
• Understanding its properties is key to studying viral oncogenesis.

Purpose of the Study:

• To purify intact pp60v-src to homogeneity.
• To characterize the enzymatic activity and stability of purified pp60v-src.

Main Methods:

• Purification involved detergent extraction and multiple chromatography steps (hydroxylapatite, butyl agarose, DEAE-Sephacel, ADP-agarose, Sephacryl S-200).
• Protein purity was assessed by SDS-PAGE and 2D gel electrophoresis.
• Enzyme activity assays were performed using alpha-casein as a substrate.

Main Results:

• Achieved over 2400-fold purification of intact pp60v-src.
• Purified pp60v-src showed qualitatively similar activities to partially purified enzyme.
• pp60v-src dephosphorylated during purification; required Nonidet P-40 and KCl to remain monomeric.
• Autophosphorylation rate was concentration-independent (intramolecular).
• Nucleotide preincubation preserved enzyme activity, while absence of nucleotides reduced it by 80%.

Conclusions:

• Nucleotide protection, not autophosphorylation, likely explains increased activity after ATP incubation.
• Purified pp60v-src provides a tool for further biochemical and structural studies.
• Findings shed light on the regulation and stability of viral tyrosine kinases.