Purification and characterization of a tyrosine-specific protein kinase of Mr 60,000 and comparison with a kinase of Mr 56,000 from rat spleen.
A tyrosine-specific protein kinase of Mr 60,000 (TK-I) was purified to near homogeneity from the particulate fraction of rat spleen. The purification procedure involved sequential chromatography of the detergent-solubilized enzyme on DEAE-Sephacel and hydroxyapatite columns. Polyacrylamide-gel electrophoresis under denaturing conditions showed one major polypeptide, of Mr 60,000. Gel filtration of the enzyme on Sephacryl S-200 column showed a single peak of kinase activity, of apparent Mr 60,000. On incubation with [gamma-32P]ATP, it showed a phosphoprotein of Mr 60,000 as a result of autophosphorylation. The autophosphorylation of the kinase occurred only at tyrosine residues. Incubation of TK-I with ATP (but not with ADP) resulted in an increase in its tyrosine-specific protein kinase activity. The time course of autophosphorylation of TK-I was very similar to the time course of activation by ATP. These and other experiments suggest that autophosphorylation might be responsible for activation of TK-I observed on incubation with ATP. A second tyrosine-specific protein kinase (TK-II) was isolated from the particulate fraction of rat spleen. A highly purified preparation of TK-II on incubation with [gamma-32P]ATP gave a major phosphoprotein, of Mr 56,000. TK-II was different from TK-I in several properties: (a) substrate specificity; (b) chromatographic behaviour; (c) phosphopeptide maps; and (d) inhibition by tosyl-lysylchloromethane. Antisera raised against TK-I did not cross-react with TK-II. These results suggest that TK-I and TK-II are distinct proteins, perhaps coded by two different genes.
Swarup
G
G
Centre for Cellular and Molecular Biology, India.
Subrahmanyam
G
G
Rema
V
V
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D013154:Spleen / Q000201:enzymology*
D007527:Isoenzymes; D008345:Manganese; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D008274:Magnesium