Characterization and purification of the solubilized pituitary adenylate-cyclase-activating polypeptide-1 receptor from porcine brain using a biotinylated ligand.
A specific receptor for the brain-gut neuropeptide pituitary adenylate-cyclase-activating polypeptide (PACAP-1 receptor) was solubilized with Chapso from porcine brain plasma membranes and purified. Binding of 125I-PACAP(1-27) to the solubilized material was reversed equipotently by unlabeled PACAP(1-27) and PACAP(1-38). Soluble receptors retained the binding affinities and specificities of the plasma membrane fraction. Scatchard analysis of equilibrium-binding data indicated the existence of a single high-affinity binding site (Kd = 0.23 nM, Bmax = 1.2 pmol/mg protein). Binding of 125I-PACAP(1-27) to solubilized receptors was not affected by guanosine nucleotides, suggesting that solubilization dissociates the PACAP-1 receptor/guanosine-nucleotide-binding protein complex. Affinity cross-linking of 125I-PACAP(1-27) to soluble 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 glycoprotein was purified 4-5-fold by lectin-adsorption chromatography on wheatgerm agglutinin immobilized on agarose. S-Biotinyl[Ala28-34, Cys35]PACAP(1-35) was synthesized, immobilized on streptavidin-coated magnetic Sepharose beads and used to further affinity-purify wheatgerm-agglutinin-eluted receptor material. This more than 6000-fold enriched PACAP-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 purification of PACAP-1 receptors, allowing further studies to be performed by protein chemistry.
Schäfer
H
H
Department of Medicine, Christian-Albrechts-University of Kiel, Germany.
Schmidt
W E
WE
D000818:Animals; D001710:Biotin; D001921:Brain / Q000378:metabolism*; D002846:Chromatography, Affinity; D003545:Cysteine / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006031:Glycosylation; D008024:Ligands; D009479:Neuropeptides / Q000378:metabolism; D011869:Radioligand Assay; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D008024:Ligands; D009479:Neuropeptides; D051236:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide; D011979:Receptors, Pituitary Hormone; D001710:Biotin; D003545:Cysteine