Detection of calcium-dependent regulatory protein binding components using ¹²⁵I-labeled calcium-dependent regulatory protein

The calcium-dependent regulatory protein (CDR) purified from bovine brain was iodinated with Na[¹²⁵I]I using the lactoperoxidase-glucose oxidase system. The iodinated protein retained its ability to stimulate the Ca²⁺-sensitive CDR-depleted cyclic nucleotide phosphodiesterase from bovine heart. Stimulation of the phosphodiesterase by ¹²⁵I-CDR was Ca²⁺-dependent and the labeled protein had a K(a) for activation of cyclic nucleotide phosphodiesterase that was 4 times greater than unmodified CDR. ¹²⁵I-CDR formed a Ca²⁺-dependent complex with the partially purified cyclic nucleotide phosphodiesterase which was detectable by autoradiography following electrophoresis of the complex on nondenaturing gels. This technique was used to detect CDR binding components in crude homogenates prepared from bovine heart and brain.