Functional properties of covalent beta-endorphin peptide/calmodulin complexes. Chlorpromazine binding and phosphodiesterase activation.
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. We found that 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. In contrast, 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, however, showed little detectable binding of the phenothiazine.(ABSTRACT TRUNCATED AT 250 WORDS)
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001921:Brain / Q000378:metabolism; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D002250:Carbon Radioisotopes; D002417:Cattle; D002746:Chlorpromazine / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D006152:Cyclic GMP / Q000378:metabolism; D004723:Endorphins / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D013552:Swine; D013737:Testis / Q000378:metabolism; D001615:beta-Endorphin
D002147:Calmodulin; D002250:Carbon Radioisotopes; D004723:Endorphins; D010446:Peptide Fragments; D001615:beta-Endorphin; D000242:Cyclic AMP; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D006152:Cyclic GMP; D002746:Chlorpromazine