Immune recognition of affinity-labelled cholecystokinin receptor.
The present study was undertaken to characterize the immune recognition of pancreatic cholecystokinin receptor by an anti-cholecystokinin antibody. Cholecystokinin receptor from pancreatic plasma membranes was photoaffinity labelled using the specific, cleavable probe 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK(25-33) [CCK(25-33) is the C-terminal nonapeptide of the 33-amino-acid form of cholecystokinin]. Labelled receptor was then solubilized and subsequently prepurified on immobilized wheat-germ agglutinin. The C-terminal-directed anti-cholecystokinin serum (8E) specifically immunoprecipitated a fraction of affinity-labelled cholecystokinin receptor which was identified at Mr 85,000 - 100,000 on SDS/PAGE. The binding affinity of antiserum 8E for covalently labelled cholecystokinin receptor was lower (Kd 0.11 +/- 0.02 nM) than for cholecystokinin (Kd 3.65 +/- 0.55 pM). The compound L364-718, an A-subtype cholecystokinin-receptor antagonist did not interfere with the immune recognition of cholecystokinin. However, the recognition of affinity-labelled cholecystokinin receptor was enhanced as a result of an increasing availability of cholecystokinin molecules. Indeed, the amount of immunoprecipitated receptor was doubled in the presence of 10 microM L364-718. This study offers the possibility of using an anti-cholecystokinin antibody for cholecystokinin-receptor purification and demonstrates that prepurified affinity-labelled cholecystokinin receptor retains A-subtype specificity.
Dufresne
M
M
Institut National de la Santé et de la Recherche Médicale Unité 151, Centre Hospitalier Universitaire Rangueil, Toulouse, France.
Poirot
S
S
Jimenez
J
J
Cuber
J C
JC
Vaysse
N
N
Fourmy
D
D
D000345:Affinity Labels; D000818:Animals; D000915:Antibody Affinity; D000918:Antibody Specificity; D002462:Cell Membrane / Q000032:analysis; D002766:Cholecystokinin / Q000031:analogs & derivatives* / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D008297:Male; D010179:Pancreas / Q000032:analysis; D010446:Peptide Fragments / Q000276:immunology*; D011233:Precipitin Tests; D051381:Rats; D011949:Receptors, Cholecystokinin / Q000276:immunology / Q000302:isolation & purification*
D000345:Affinity Labels; D007106:Immune Sera; D010446:Peptide Fragments; D011949:Receptors, Cholecystokinin; C061851:cholecystokinin (25-33),2-(4-azidosalicylamido)-1,3-dithiopropionate(Thr(28)-AHX(31))-; D002766:Cholecystokinin