Functional properties of covalent .beta.-endorphin peptide/calmodulin complexes. Chlorpromazine binding and phosphodiesterase activation

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Abstract
The 31-residue neuropeptide porcine .beta.-endorphin was shown to inhibit the Ca2+-dependent calmodulin activation of highly purified bovine brain cyclic nucleotide phosphodiesterase (3'', 5''-cyclic AMP 5''-nucleotidohydrolase, EC 3.1.4.17). Using a series of deletion peptides, the minimal inhibitory peptide sequence was found to correspond to .beta.-endorphin residues 14-25, confirming previously reported results for crude enzyme preparations. A correlation was found between the relative inhibitory potency of a particular .beta.-endorphin deletion peptide and the efficacy of cross-linking that peptide to calmodulin with bis(sulfosuccinimidyl) suberate, strongly implicating peptide binding to calmodulin as the mechanism of the observed inhibition. Relatively modest concentrations of chlorpromazine significantly reduced the efficiency of cross-linking .beta.-endorphin 14-31 to calmodulin. Chlorpromazine-Sepharose affinity chromatography of peptide/calmodulin adducts showed that a significant portion of the cross-linked .beta.-endorphin 14-31/calmodulin complex (stoichiometry of 1 mol/mol) retained the ability to interact with the immobilized phenothiazine in a Ca2+-dependent and calmodulin-displaceable manner. The 2:1 (peptide:protein) product exhibited no affinity for the immobilized phenothiazine. The use of this affinity chromatographic step allowed preparation of homogeneous populations of both 1:1 and 2:1 .beta.-endorphin 13-31/calmodulin complexes and assessment of their functional characteristics. Equilibrium binding studies with chlorpromazine revealed that the covalent attachment of one peptide molecule to calmodulin perturbed all phases of Ca2+-dependent drug binding, but the adduct still bound significant quantities of chlorpromazine. The 2:1 complex showed little detectable binding of the phenothiazine. .beta.-Endorphin-derived peptides and chlorpromazine binding domain(s) on calmodulin possess some commonality. At least a portion of these inhibitory molecule domain(s) seems intimately involved in phosphodiesterase recognition and activation as both the 1:1 and 2:1 complexes exhibited negligible ability to activate or inhibit the calmodulin-dependent activation of the enzyme.

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