Purification and characterization of the feline sarcoma virus tyrosine-specific kinase pp85gag-fes.
The transforming phosphoprotein pp85gag-fes of the Snyder-Theilen strain of feline sarcoma virus (ST-FeSV) was purified in a form which exhibits tyrosine-specific kinase activity. Cell lysates of ST-FeSV-transformed mink nonproducer cells were applied to a column of DEAE-Sephacel and eluted with a linear gradient of NaCl in phosphate buffer. Kinase activity was found uncomplexed (pp85gag-fes) in the flow-through and was eluted with 200 mM NaCl in a complex with pp50 and pp90. The flow-through material was further fractionated by chromatography on hydroxylapatite, followed by phosphocellulose and a final gel filtration step using Sephacryl S200. The final preparation was specifically enriched 1400-fold, and tyrosine-specific kinase activity was increased by about 300-fold as determined by autophosphorylation or phosphorylation of casein. Pp85gag-fes kinase activity was inhibited by nicotinamide adenosine dinucleotide (NAD) and slightly inhibited by NADH, while quercetin, a strong inhibitor for pp60src-associated tyrosine kinase activity, had no inhibiting effect.
D000818:Animals; D002364:Caseins / Q000378:metabolism; D002460:Cell Line; D002845:Chromatography; D015683:Gene Products, gag; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D008907:Mink; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D051381:Rats; D012190:Retroviridae / Q000201:enzymology*; D012191:Retroviridae Proteins / Q000302:isolation & purification* / Q000378:metabolism; D005253:Sarcoma Viruses, Feline / Q000201:enzymology*; D015032:Zinc / Q000378:metabolism
D002364:Caseins; D015683:Gene Products, gag; D012191:Retroviridae Proteins; D008345:Manganese; D011505:Protein-Tyrosine Kinases; D008274:Magnesium; D015032:Zinc