Separation of calmodulin from calcium-activated protein kinase using calcium-dependent hydrophobic interaction chromatography.

To determine whether a Ca2+-activated protein kinase is regulated by calmodulin, it is necessary to separate it from endogenous calmodulin and from protein kinase activity that is not calcium dependent. We describe here a procedure for achieving these goals using Ca2+-dependent hydrophobic interaction chromatography on phenyl Sepharose in combination with a pH change. The procedure is based on the observation that while calmodulin solubilized from apple fruit membranes binds to phenyl Sepharose in a Ca2+-dependent fashion at both pH 7.0 and 8.5, Ca2+-activated protein kinase from the same source only shows a Ca2+-dependent interaction above pH 7.5. The implications of this finding for the regulation of this Ca2+-activated protein kinase are briefly discussed.