DETERMINATION OF BINDING PARAMETERS OF CYCLIC-AMP AND ITS ANALOGS TO CYCLIC AMP-DEPENDENT PROTEIN-KINASE BY THE FLUORESCENT-PROBE METHOD

The method for determination of dissociation constants for cyclic AMP and its analogs bound to cyclic AMP-dependent protein kinase from pig brain is described. The technique for measuring the binding parameters of the ligands is based on the changes in the fluorescent spectrum of etheno cyclic AMP once it is bound to protein kinase. The dissociation constants for a number of non-fluorescent cyclic AMP analogs were determined in the competitive displacement of etheno cyclic AMP by these analogs. The number of cyclic AMP-binding sites in the pig brain protein kinase was found to be 2.2; no cooperativity was observed upon binding. The holoenzyme complex (Mr = 180 000) of the protein kinase under study was established to have the stoichiometry of R2C2 type under native conditions. © 1979.