Summary
The pituitary adenylate cyclase-activating polypeptide (PACAP), including PACAP27 and PACAP38, stimulates adenylate cyclase via specific PACAP receptors that are coupled to G proteins. The present study describes the application of avidin-biotin technology to the purification of the PACAP receptor from bovine brain membranes. The PACAP receptor was solubilized from the membranes with digitonin and partially purified by DEAE-Toyopearl and hydroxylapatite chromatography for removal of avidin-binding proteins. The partially purified PACAP receptor was mixed with a biotinylated ligand (PACAP27Cys(biotin)-NH2) to form a receptor-ligand complex, adsorbed onto avidinagarose, and then eluted with 1.0 M NaCl-acetate buffer (pH 4.0). The affinity-purified receptor was further purified by hydroxylapatite and gel-filtration chromatography. Analysis of the purified preparation by SDS-polyacrylamide gel electrophoresis and silver staining showed a single broad protein band with an M r = 55 000 to 60 000. The specific activity of the purified receptor (17.2 nmol/mg) was close to the theoretical value. The dissociation constant (K d = 25.8 pM) was similar to that of the crude solubilized PACAP receptor. Thus, the PACAP receptor was purified to near homogeneity in a fully active form preserving high affinity for PACAP. The present study demonstrates that affinity chromatography using biotinylated ligands is a promising method for the purification of the PACAP receptor.
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© 1996 Birkhäuser Verlag
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Ohtaki, T., Kitada, C., Onda, H. (1996). Purification of the Receptor for Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) using Biotinylated Ligands. In: Meier, T., Fahrenholz, F. (eds) A Laboratory Guide to Biotin-Labeling in Biomolecule Analysis. BioMethods. Birkhäuser Basel. https://doi.org/10.1007/978-3-0348-7349-9_3
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DOI: https://doi.org/10.1007/978-3-0348-7349-9_3
Publisher Name: Birkhäuser Basel
Print ISBN: 978-3-0348-7351-2
Online ISBN: 978-3-0348-7349-9
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