Mills A, Demoliou-Mason C D, Barnard E A
Medical Research Council Molecular Neurobiology Unit, Medical Research Council Centre, Cambridge, Great Britain.
J Biol Chem. 1988 Jan 5;263(1):13-6.
The neurotensin receptor protein, solubilized with digitonin/asolectin from bovine cerebral cortex membranes, was purified to apparent homogeneity by affinity chromatography using immobilized neurotensin. The product exhibits saturable and specific binding of [3,11-tyrosyl-3,5-3H]neurotensin with an apparent affinity (Kd = 5.5 nM) comparable to that measured in intact membranes and crude soluble extracts. The affinity-purified material, after reduction with 100 mM dithiothreitol, in denaturing gel electrophoresis showed a single polypeptide of Mr 72,000. Under nonreducing conditions the apparent Mr, however, was 50,000, suggesting the presence of intramolecular disulfide bonds. The purified neurotensin receptor was judged to be homogeneous, in that (i) only a single polypeptide was detectable; and (ii) the overall purification was 30,000-50,000-fold, giving a specific neurotensin-binding activity close to the theoretical maximum.
用洋地黄皂苷/大豆卵磷脂从牛大脑皮层膜中增溶得到的神经降压素受体蛋白，通过使用固定化神经降压素的亲和层析法纯化至表观均一。该产物对[3,11-酪氨酰-3,5-³H]神经降压素表现出饱和且特异的结合，其表观亲和力（Kd = 5.5 nM）与在完整膜和粗可溶性提取物中测得的相当。经100 mM二硫苏糖醇还原后的亲和纯化物质，在变性凝胶电泳中显示出一条Mr为72,000的单一多肽。然而，在非还原条件下，表观Mr为50,000，表明存在分子内二硫键。纯化的神经降压素受体被判定为均一，因为（i）仅可检测到一条单一多肽；（ii）总体纯化倍数为30,000 - 50,000倍，产生的特异神经降压素结合活性接近理论最大值。
