MEASUREMENT OF THE PROTEIN-TYROSINE KINASE-ACTIVITY OF C-SRC USING TIME-RESOLVED FLUOROMETRY OF EUROPIUM CHELATES

A nonradioactive, sensitive assay method to evaluate the activity of p rotein tyrosine kinases is described. This method utilizes europium ch elate-labeled anti-phosphotyrosine antibodies to detect phosphate tran sfer to a polymeric substrate coated onto microtiter plate wells. The amount of phosphorylation is then detected using time-resolved, dissoc iation-enhanced fluorescence, Recombinant c-src was used to demonstrat e that substrate phosphorylation was dependent on incubation time, enz yme concentration, and the amount of substrate used to coat the microt iter plate wells. A series of proprietary c-src inhibitors was evaluat ed in competition assays, and demonstrated a rank order of potency whi ch was identical to that determined by other assay methods. Substrate phosphorylation was also demonstrated to be dependent on the concentra tion of ATP present during the kinase reaction, Because the kinase ass ay can be performed with different ATP concentrations (unlike with ass ays utilizing radioactive ATP analogs), the assay described can be use d to distinguish compounds that compete for the ATP or substrate bindi ng sites of the kinase. (C) 1996 Academic Press, Inc.