Dufresne M, Poirot S, Jimenez J, Cuber J C, Vaysse N, Fourmy D
Institut National de la Santé et de la Recherche Médicale Unité 151, Centre Hospitalier Universitaire Rangueil, Toulouse, France.
Eur J Biochem. 1990 Jul 20;191(1):141-6. doi: 10.1111/j.1432-1033.1990.tb19103.x.
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.
本研究旨在通过抗胆囊收缩素抗体来表征胰腺胆囊收缩素受体的免疫识别。使用特异性可裂解探针125I标记的2-(对叠氮水杨酰胺基)-1,3-二硫代丙酸-[苏氨酸28，Ahx31]CCK(25-33)[CCK(25-33)是33个氨基酸形式的胆囊收缩素的C末端九肽]对胰腺质膜中的胆囊收缩素受体进行光亲和标记。然后将标记的受体溶解，并随后在固定化麦胚凝集素上进行预纯化。C末端导向的抗胆囊收缩素血清(8E)特异性免疫沉淀了一部分亲和标记的胆囊收缩素受体，该受体在SDS/PAGE上的分子量为85,000 - 100,000处被鉴定。抗血清8E对共价标记的胆囊收缩素受体的结合亲和力(解离常数Kd为0.11±0.02 nM)低于对胆囊收缩素的结合亲和力(解离常数Kd为3.65±0.55 pM)。化合物L364-718，一种A亚型胆囊收缩素受体拮抗剂，不干扰胆囊收缩素的免疫识别。然而，由于胆囊收缩素分子可用性的增加，亲和标记的胆囊收缩素受体的识别得到增强。事实上，在存在10 microM L364-718的情况下，免疫沉淀的受体量增加了一倍。本研究提供了使用抗胆囊收缩素抗体进行胆囊收缩素受体纯化的可能性，并证明预纯化的亲和标记的胆囊收缩素受体保留了A亚型特异性。
