Voisin T, Couvineau A, Rouyer-Fessard C, Laburthe M
Unité de Recherche sur la Différenciation et la Neuroendocrinologie de Cellules Digestives, Institut National de la Santé et de la Recherche Médicale, Villejuif, France.
J Biol Chem. 1991 Jun 15;266(17):10762-7.
Peptide YY (PYY) receptors were solubilized from rat jejunal crypts using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonic acid (CHAPS). The binding of [125I-Tyr36]monoiodo-PYY ([125I]PYY) to CHAPS extracts was time-dependent and reversible. The order of potency of PYY-related peptides for inhibiting [125I]PYY binding was PYY greater than neuropeptide Y much greater than pancreatic polypeptide. Scatchard analysis of equilibrium binding data indicated the presence in soluble extracts of a single class of binding sites with a Kd of 1.02 +/- 0.26 nM and a Bmax of 79 +/- 6 fmol/mg protein. Gel filtration on Sephacryl S-300 and ultracentrifugation on sucrose density gradients of soluble [125I] PYY-receptor complexes revealed a single binding component with the following hydrodynamic parameters: Stokes radius, 4.43 nm; s20,w, 2.48 S; Mr, 48,000; frictional ratio, 1.82. Solubilized PYY receptors bound specifically to concanavalin A-, wheat germ agglutinin-, and soybean-coupled Sepharose, supporting their glycoproteic nature. After cross-linking with disuccinimidyl suberate, electrophoresis of covalent [125I]PYY-receptor complexes in membranes or CHAPS extracts revealed the presence of two bands of Mr 49,000 or 28,000 whose labeling was completely abolished by 1 microM unlabeled PYY. The Mr 49,000 band probably corresponded to the Mr 48,000 PYY-receptor complex evidenced by hydrodynamic studies. Assuming one molecule of [125I]PYY (Mr 4,000) was bound per molecule of receptor, these data show that intestinal PYY receptor consists of a Mr 44,000 glycoprotein after solubilization with CHAPS. The availability of this CHAPS-soluble receptor from rat jejunum represents a major step toward the purification of this newly characterized receptor.
使用3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸(CHAPS)从大鼠空肠隐窝中溶解肽YY(PYY)受体。[125I-Tyr36]单碘-PYY([125I]PYY)与CHAPS提取物的结合具有时间依赖性且可逆。抑制[125I]PYY结合的PYY相关肽的效力顺序为：PYY大于神经肽Y远大于胰多肽。对平衡结合数据的Scatchard分析表明，可溶性提取物中存在一类结合位点，其解离常数(Kd)为1.02±0.26 nM，最大结合容量(Bmax)为79±6 fmol/mg蛋白质。在Sephacryl S-300上进行凝胶过滤以及对可溶性[125I]PYY-受体复合物进行蔗糖密度梯度超速离心，结果显示单一结合成分具有以下流体动力学参数：斯托克斯半径为4.43 nm；沉降系数(s20,w)为2.48 S；相对分子质量(Mr)为48,000；摩擦系数为1.82。溶解的PYY受体与伴刀豆球蛋白A、麦胚凝集素和大豆偶联的琼脂糖特异性结合，证明其糖蛋白性质。用辛二酸二琥珀酰亚胺酯交联后，对膜或CHAPS提取物中的共价[125I]PYY-受体复合物进行电泳，结果显示存在两条相对分子质量分别为49,000或28,000的条带，其标记被1 μM未标记的PYY完全消除。相对分子质量49,000的条带可能对应于流体动力学研究证明的相对分子质量48,000的PYY-受体复合物。假设每个受体分子结合一个[125I]PYY分子(相对分子质量4,000)，这些数据表明，用CHAPS溶解后，肠道PYY受体由相对分子质量44,000的糖蛋白组成。从大鼠空肠中获得这种CHAPS可溶性受体是朝着纯化这种新鉴定的受体迈出的重要一步。
