Solid-phase tyrosine-specific protein kinase assay in multiwell substrate-immobilized polyacrylamide gel.
Since tyrosine-specific protein kinase (TPK) is much less abundant than Ser/Thr-specific kinases in cells, determination of TPK activity in crude cell extracts or column chromatography eluates has been difficult. This is compounded by the absence of a rapid, economical method for the separation of high endogenous protein phosphorylation background from exogenously added tyrosine-containing substrates. We have developed a new solid-phase assay, which provides high sensitivity and efficiency at a low cost for assaying the TPK activity of crude enzyme preparations. This assay utilizes immobilized tyrosine-containing synthetic polymers such as (Glu:Tyr, 4:1)n in polyacrylamide gels. The kinase reaction is started by adding crude enzyme solutions and [tau-32P]ATP-metal ion mixtures into microtiter-size wells made in the gels. After the phosphorylation reaction, the reaction mixtures are removed and the gels are prewashed in water followed by electrophoresis to completely remove free radioactive ATP. 32P incorporation into the immobilized TPK-specific substrate can be detected by autoradiography and quantitated by cutting the gel pieces and counting them with a liquid scintillation counter. The simple, rapid method should facilitate screening of TPK inhibitors and activators as well as examining the substrate specificity of TPKs. Other enzymes, including Ser/Thr-specific protein kinases, can also be analyzed by this technique.
Sahal
D
D
Beckman Research Institute of the City of Hope, Department of Molecular Genetics, Duarte, California 91010-0269.
Fujita-Yamaguchi
Y
Y
D000180:Acrylic Resins; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002845:Chromatography / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004800:Enzymes, Immobilized; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008832:Microchemistry / Q000379:methods; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000032:analysis* / Q000378:metabolism; D013379:Substrate Specificity
D000180:Acrylic Resins; D004800:Enzymes, Immobilized; D010761:Phosphorus Radioisotopes; C016680:polyacrylamide gels; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases