Purification of the heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase by affinity chromatography

Abstract

The recently discovered heat-stable inhibitor protein of the Ca2+-activated cyclic nucleotide phosphodiesterase was purified 238, 214-fold from bovine brain extract using an affinity column of the modulator protein-Sepharose 4B conjugate. The purified sample appears to be homogeneous as judged by sodium dodecyl sulfate gel electrophoresis. The protein band has a mobility corresponding to that of a polypeptide of MW 68,000. Since the heat-stable inhibitor protein has a MW of 70,000 under nondenaturing conditions, it suggests that it is a monomeric protein. The protein has no inhibitory activity toward the c[cyclic]AMP-dependent protein kinase or protein phosphatase. The purified sample was tested for various enzyme activities which include ATPase, GTPase, cAMP phosphodiesterase, cGMP phosphodiesterase, 5''-nucleotidase and protein kinase. None of these activities are exhibited by the purified sample.