CHARACTERIZATION AND PURIFICATION OF THE SOLUBILIZED PITUITARY ADENYLATE-CYCLASE-ACTIVATING POLYPEPTIDE-1 RECEPTOR FROM PORCINE BRAIN USINGA BIOTINYLATED LIGAND

A specific receptor for the brain-gut neuropeptide pituitary adenylate -cyclase-activating polypeptide (PACAP-1 receptor) was solubilized wit h Chapso from porcine brain plasma membranes and purified. Binding of I-125-PACAP(1-27) to the solubilized material was reversed equipotentl y by unlabeled PACAP(1-27) and PACAP(1-38). Soluble receptors retained the binding affinities and specificities of die plasma membrane fract ion. Scatchard analysis of equilibrium-binding data indicated the exis tence of a single high-affinity binding site (K(d) = 0.23 nM, Bmax = 1 .2 pmol/mg protein). Binding of I-125-PACAP(1-27) to solubilized recep tors was not affected by guanosine nucleotides, suggesting that solubi lization dissociates the PACAP-1 receptor/guanosine-nucleotide-binding protein complex. Affinity cross-linking of I-125-PACAP(1-27) to solub le PACAP-1 receptors identified a specifically labeled 60-kDa protein. Enzymic deglycosylation of soluble affinity-labeled receptors reduced the apparent molecular mass by 10 kDa. The solubilized receptor glyco protein was purified 4-5-fold by lectin-adsorption chromatography on w heatgerm agglutinin immobilized on agarose. S-Biotinyl[Ala28-34, Cys35 ]PACAP(I-35) was synthesized, immobilized on streptavidin-coated magne tic Sepharose beads and used to further affinity-purify wheatgerm-aggl utinin-eluted receptor material. This more than 6000-fold enriched PAC AP-1-receptor-preparation retained single-class high-affinity binding and consisted of an almost homogenous 55-60-kDa protein identified by silver staining. In conclusion, we established a rapid method for puri fication of PACAP-1 receptors, allowing further studies to be performe d by protein chemistry.