Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography.
Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.
Couvineau
A
A
Unité de Recherche sur la Différenciation et la Neuroendocrinologie de Cellules Digestives, Institut National de la Santé et de la Recherche Médicale U178, Villejuif, France.
Voisin
T
T
Guijarro
L
L
Laburthe
M
M
D000818:Animals; D002793:Cholic Acids; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D007700:Kinetics; D008099:Liver / Q000378:metabolism*; D008970:Molecular Weight; D011964:Receptors, Gastrointestinal Hormone / Q000302:isolation & purification* / Q000378:metabolism; D018005:Receptors, Vasoactive Intestinal Peptide; D013552:Swine; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism
D002793:Cholic Acids; D007202:Indicators and Reagents; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate