Purification and characterization of a protein tyrosine kinase from bovine spleen.
Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006657:Histones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D008345:Manganese / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000302:isolation & purification*; D013154:Spleen / Q000201:enzymology*
D002364:Caseins; D006657:Histones; D008345:Manganese; D009532:Nickel; D011505:Protein-Tyrosine Kinases; D008274:Magnesium; D002118:Calcium