Characterization and purification of the solubilized pituitary adenylate-cyclase-activating polypeptide-1 receptor from porcine brain using a biotinylated ligand

Eur J Biochem. 1993 Nov 1;217(3):823-30. doi: 10.1111/j.1432-1033.1993.tb18310.x.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biotin
  • Brain / metabolism*
  • Chromatography, Affinity
  • Cysteine / analysis
  • Electrophoresis, Polyacrylamide Gel
  • Glycosylation
  • Ligands
  • Neuropeptides / metabolism
  • Radioligand Assay
  • Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide
  • Receptors, Pituitary Hormone / chemistry
  • Receptors, Pituitary Hormone / isolation & purification
  • Receptors, Pituitary Hormone / metabolism*
  • Swine

Substances

  • Ligands
  • Neuropeptides
  • Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide
  • Receptors, Pituitary Hormone
  • Biotin
  • Cysteine