Kinetic Characterization of p34cdc2/Cyclin B Kinase-Mediated Phosphorylation of Peptides Derived from Histone H1 Using Phosphocellulose Filter Binding and Scintillation Proximity Assays

Activation of p34cdc2 requires a complex series of protein/protein interactions and specific phosphorylation and de-phosphorylation events to occur. The cellular role of p34cdc2 kinase lies in the regulation of eukaryotic cell entry into mitosis, in particular into the G2/M transition. This study examines the kinetic characterization of p34cdc2/cyclin B kinase utilizing both the phosphocellulose filter binding assay (FBA) and a modified version of the scintillation proximity assay (SPA). Several factors were identified that elicited an effect on the kinetic constants determined for the phosphorylation reaction, with emphasis being placed on the Km apparent (Kmapp) values. Factors identified included the concentration of adenosine triphosphate (ATP) used in the reaction, the addition of a biotin label to the peptide substrate as required for capture of the phospholabeled peptide by SPA, and the source from which the p34cdc2/cyclin B kinase was isolated. The Kmapp is the kinetic constant most frequently rep...