Solid-phase protein-tyrosine kinase assay.

Publisher Summary This chapter presents a new solid-phase tyrosine-specific protein kinase (TPK) assay to measure TPK activity of crude enzyme preparations. The phosphorylation reaction is performed in a well of a polyacrylamide gel into which a TPK specific polymeric substrate such as (Glu,Tyr) n , 4:1 has been immobilized by copolymerization. Incorporation of 32 P into the immobilized TPK-specific substrate is detected by either autoradiography or liquid scintillation counting of the gel pieces excised from the bottom of the well. The major advantages of the solid-phase TPK assay are that multiple assays can be conveniently performed and that TPK activity can be detected in crude enzyme preparations without interference by serine/threonine (Ser/Thr) kinases whose catalytic activity accounts for over 99% of cellular protein phosphorylation. The solid-phase TPK assay can be useful for screening inhibitors and activators of TPKs as well as examining substrate specificity. The solid-phase assay may also be useful for assaying protein phosphatases since the product of the protein kinase reaction in the gels can be used as substrates for the protein phosphatase reaction. The disadvantage of the solid-phase assay is low sensitivity due to the limited accessibility of the substrate to the enzymes.