Identification of .beta.-endorphin residues 14-25 as a region involved in the inhibition of calmodulin-stimulated phosphodiesterase activity

Abstract

The inhibition of the calmodulin-mediated stimulation of bovine brain cyclic nucleotide phosphodiesterase activity (cAMP 5''-nucleotidohydrolase, EC 3.1.4.17) by the 31-residue opiate peptide .beta.-endorphin was investigated. Using conditions in which porcine brain calmodulin (6 nM) is limiting (i.e., to give a 3-fold, Ca2+-dependent stimulation of enzymic activity toward cGMP), the domain of .beta.-endorphin responsible for the inhibition was mapped by using a series of deletion peptides. .beta.-Endorphin exhibited an ED50 of several micromolar under the conditions employed, and several amino-terminal deletion peptides were essentially as inhibitory as the parent peptide. Methionine enkephalin and various carboxy-terminal deletion peptides had no demonstrable effect at concentrations of 100-200 .mu.M. Peptides 1-25 and 1-27 (C'' fragment) inhibited the calmodulin-dependent activity of phosphodiesterase, but higher concentrations were required than of .beta.-endorphin. Studies using combined amino- and carboxy-terminal deletion peptides demonstrated that peptide 14-25 was the shortest peptide examined that was capable of inhibiting calmodulin stimulation of phosphodiesterase activity under the conditions used. There was no evidence to indicate that the amino-terminal region comprising residues 1-13 of .beta.-endorphin contributes to the measured inhibition of calmodulin-stimulated enzymic activity. The circular dichroic spectra of calmodulin, .beta.-endorphin and mixtures of the 2 were obtained, and the ellipticity of the peptide-protein mixtures at 221 nm exceeded that expected by assuming simple additivity. This finding is consistent with a direct interaction of .beta.-endorphin with calmodulin which seems to lead to enhanced helicity of one or both components. Using a spin-labeled calmodulin derivative, .beta.-endorphin had only subtle effects on the electron paramagnetic resonance spectrum. This finding, in conjunction with the circular dichroic spectra, suggests that the peptides do not act by inducing a calmodulin conformation like that in the Ca2+-free state. The .beta.-endorphin-mediated inhibition of calmodulin-dependent phosphodiesterase activity arises from a nonopiate portion of the molecule. Moreover, 2 naturally occurring .beta.-endorphin-derived peptides, des-Tyr1-.beta.-endorphin and C'' fragment, are inhibitory while others, .alpha.- and .gamma.-endorphin, are not.