Purification of A-subtype pancreatic cholecystokinin receptor by immunoaffinity chromatography.
So far, no efficient affinity chromatography for CCK receptor purification has been reported that prevented obtention of sequenceable amounts of purified receptor. In this work, 10% of plasma membrane receptor sites were specifically cross-linked with the photoreactive cleavable agonist 125I-ASD-[Thr28, Ahx31]-CCK-25-33, solubilized by NP-40, chromatographied on immobilized wheat germ agglutinin and further immunopurified using anti-CCK antibodies to an overall rate of 3000-3600-fold. Analysis of eluted material demonstrated a protein migrating at Mr 85,000-100,000 and the absence of 35S-labeled impurity. This single and efficient affinity chromatography should provide enough homogeneous receptor protein for microsequence determination and leads to consider immunoaffinity chromatography on immobilized anti-ligand antibodies as a potential tool for purification of membrane receptors.
Dufresne
M
M
INSERM U151, CHU Rangueil, Toulouse, France.
Poirot
S
S
Jimenez
J
J
Vaysse
N
N
Fourmy
D
D
D000345:Affinity Labels; D000818:Animals; D002462:Cell Membrane / Q000737:chemistry; D002766:Cholecystokinin / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D007158:Immunologic Techniques; D008970:Molecular Weight; D017830:Octoxynol; D010179:Pancreas / Q000737:chemistry*; D010777:Photochemistry; D011092:Polyethylene Glycols; D051381:Rats; D012995:Solubility; D014909:Wheat Germ Agglutinins
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D014909:Wheat Germ Agglutinins; D011092:Polyethylene Glycols; D017830:Octoxynol; D002766:Cholecystokinin; C010615:Nonidet P-40