Fourmy D, Lopez P, Poirot S, Jimenez J, Dufresne M, Moroder L, Powers S P, Vaysse N
Groupe de Recherche de Biologie et Pathologie Digestine, INSERM U 151, CHU Rangueil, Toulouse, France.
Eur J Biochem. 1989 Nov 6;185(2):397-403. doi: 10.1111/j.1432-1033.1989.tb15128.x.
Biochemical studies on receptors for peptides are most often carried out on affinity-labelled (peptide-receptor) complexes. Necessarily, the assumption is made that a covalent (peptide-receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity-labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity-labelling probe in order to minimize structural modifications occurring within the affinity-labelled cholecystokinin (CCK) receptor protein. The probe was 125I-labelled 2-(p-azidosalicylamido)-1,3-dithiopropionate-[Thr28,Ahx31 ]CCK-25-33, (125I-ASD-[Thr28,Ahx31]CCK-25-33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK-25-33 via its N-terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I-ASD-[Thr28,Ahx31]CCK-25-33 was time- and temperature-dependent and reversible. At 25 degrees C, a steady-state level was reached after 60 min and half-maximal dissociation after 38 min. Binding was inhibited by [Thr28,Ahx31]CCK-25-33 and L-364-718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I-ASD-[Thr28,Ahx31]CCK-25-33 identified a glycoprotein of Mr 85,000-100,000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu-C generated a main fragment of Mr 30,000-34,000. The same glycoprotein was photoaffinity labelled with 125I-DTyr-Gly-[Ahx28,31,pNO2Phe33]CCK-26-33 (Ahx, 2-aminohexanoic acid; pNO2Phe,p-nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I-ASD-[Thr28,Ahx31]CCK-25-33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.
对肽类受体的生化研究大多是在亲和标记的（肽 - 受体）复合物上进行的。必然地，人们假定共价（肽 - 受体）复合物的行为与天然受体相同。这一假设的有效性取决于亲和标记技术以及用于生化表征的分析方法的分辨率。我们设计了一种新的亲和标记探针，以尽量减少亲和标记的胆囊收缩素（CCK）受体蛋白内发生的结构修饰。该探针是125I标记的2 - （对 - 叠氮水杨酰胺基）-1,3 - 二硫代丙酸 - [苏氨酸28，α - 氨基己酸31]CCK - 25 - 33，（125I - ASD - [苏氨酸28，α - 氨基己酸31]CCK - 25 - 33），其肽部分通过还原从其结合位点释放。它是通过将一种光活化化学物质经由其N端与[苏氨酸28，α - 氨基己酸31]CCK - 25 - 33偶联而获得的。所得肽经高效液相色谱纯化，并在氯胺T存在下进行放射性碘化。[125I - ASD - [苏氨酸28，α - 氨基己酸31]CCK - 25 - 33的结合具有时间和温度依赖性且是可逆的。在25℃时，60分钟后达到稳态水平，38分钟后达到半数最大解离。[苏氨酸28，α - 氨基己酸31]CCK - 25 - 33和L - 364 - 718拮抗剂分别以IC50为0.4 nM和0.9 nM抑制结合。125I - ASD - [苏氨酸28，α - 氨基己酸31]CCK - 25 - 33对胰腺质膜的光亲和标记鉴定出一种分子量为85,000 - 100,000的糖蛋白，该糖蛋白保留在固定化的麦胚凝集素上。内肽酶Glu - C的酶切产生了一个分子量为30,000 - 34,000的主要片段。相同的糖蛋白用125I - DTyr - Gly - [α - 氨基己酸28,31，对硝基苯丙氨酸33]CCK - 26 - 33（α - 氨基己酸，2 - 氨基己酸；对硝基苯丙氨酸，对 - 硝基苯丙氨酸）进行光亲和标记，这是一种其光不稳定基团位于胆囊收缩素结合域的内源性探针。125I - ASD - [苏氨酸28，α - 氨基己酸31]CCK - 25 - 33是用于生物学和生物化学研究胆囊收缩素受体的一种潜在有力工具。
