DETECTION OF CALMODULIN-BINDING PROTEINS USING A P-32 LABELED GST-CALMODULIN FUSION PROTEIN AND A NOVEL RENATURATION PROTOCOL

To identify calmodulin-binding proteins in cellular extracts and tissu e homogenates and to analyze purified calmodulin target proteins, over lay procedures using I-125-calmodulin or more recently, nonradioactive biotinylated calmodulin have been widely used. Here we describe a rap id, alternative method for detecting calmodulin-binding proteins with a P-32-labeled calmodulin probe generated as a glutathione-S-transfera se (GST)-fusion protein. We used a modified pGEX-2TK vector which cont ains the flag epitope and the consensus sequence RR-A-S, that can be p hosphorylated by the cAMP-dependent protein kinase A. The fusion prote in is easily purified from bacterial lysates by affinity chromatograph y using glutathione-Sepharose(R) 4B beads. Phosphorylation of GST-calm odulin is performed directly on the beads and, after elution with redu ced glutathione, the labeled calmodulin probe can be used for overlay experiments. We also describe a rapid renaturation protocol that enhan ces the signal for some but not all calmodulin-binding proteins and is used after the proteins have been transferred to nitrocellulose filte rs. Furthermore, we have compared the specificity and sensitivity of t he P-32-labeled GST-calmodulin overlay with those of (125)1-calmodulin and biotinylated calmodulin, clearly indicating that our newly develo ped protocol is a suitable alternative to conventionally used calmodul in overlay procedures.