KINETIC CHARACTERIZATION OF P34(CDC2) CYCLIN-B KINASE-MEDIATED PHOSPHORYLATION OF PEPTIDES DERIVED FROM HISTONE H1 USING PHOSPHOCELLULOSE FILTER BINDING AND SCINTILLATION PROXIMITY ASSAYS/

Activation of p(34cdc2) requires a complex series of protein/protein i nteractions and specific phosphorylation and dephosphorylation events to occur, The cellular role of p(34cdc2) kinase lies in the regulation of eukaryotic cell entry into mitosis, in particular into the G(2)/M transition, This study examines the kinetic characterization of p(34cd c2)/cyclin B kinase utilizing both the phosphocellulose filter binding assay (FBA) and a modified version of the scintillation proximity ass ay (SPA), Several factors were identified that elicited an effect on t he kinetic constants determined for the phosphorylation reaction, with emphasis being placed on the K-m, apparent (K-mapp) values, Factors i dentified included the concentration of adenosine triphosphate (ATP) u sed in the reaction, the addition of a biotin label to the peptide sub strate as required for capture of the phospholabeled peptide by SPA, a nd the source from which the p(34cdc2)/cyclin B kinase was isolated, T he K-mapp is the kinetic constant most frequently reported in the exam ination of protein phosphorylation reactions, generally being derived from simple Lineweaver-Burk analysis, In a two-substrate reaction, how ever, the K-mapp may not be the most informative constant, as it will be influenced by the concentration of the second substrate. In this st udy, true K-m values were determined for ATP and a biotinylated peptid e substrate used in the p(34cdc2) kinase-mediated phosphorylation reac tion, Alberty and Dalziel constants were derived from secondary Linewe aver-Burk analysis of the phosphocellulose filter binding and SPA data , The kinetic constants determined by filter binding and scintillation proximity displayed good correlation, thus confirming the utility of scintillation proximity for the purpose of enzyme kinetic studies.