Mazella J, Chabry J, Kitabgi P, Vincent J P
Centre de Biochimie, Université de Nice, Faculté des Sciences, France.
J Biol Chem. 1988 Jan 5;263(1):144-9.
Neurotensin receptors were solubilized from mouse brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). The binding of 125I-labeled [Tyr3]neurotensin to the soluble fraction was time-dependent, saturable, and reversible. Unlabeled neurotensin and its analogues acetylneurotensin (8-13), neurotensin (9-13), and neurotensin (1-12) competitively antagonized the binding of 125I-labeled [Tyr3]neurotensin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane-bound receptors. Scatchard analysis of equilibrium binding data indicated that the soluble extract contained a single class of neurotensin binding sites with a Kd of 0.36 nM and a Bm of 63 fmol/mg. As already observed with membrane-bound receptors, the affinity of neurotensin for the soluble binding activity was decreased by Na+ ions. By contrast, soluble receptors were no longer sensitive to GTP and the antihistamine drug levocabastine. A molecular weight of about 100,000 was determined for soluble neurotensin receptors both under native conditions by gel filtration on Ultrogel AcA 34 and under denaturating conditions by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling.
使用两性离子去污剂3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸（CHAPS）从小鼠脑中溶解神经降压素受体。125I标记的[酪氨酸3]神经降压素与可溶性部分的结合具有时间依赖性、饱和性和可逆性。未标记的神经降压素及其类似物乙酰神经降压素（8-13）、神经降压素（9-13）和神经降压素（1-12）竞争性拮抗125I标记的[酪氨酸3]神经降压素与CHAPS溶解提取物的结合，其相对效力与膜结合受体观察到的相似。对平衡结合数据的Scatchard分析表明，可溶性提取物含有一类单一的神经降压素结合位点，Kd为0.36 nM，Bm为63 fmol/mg。正如在膜结合受体中已经观察到的那样，神经降压素对可溶性结合活性的亲和力被Na+离子降低。相比之下，可溶性受体不再对GTP和抗组胺药物左卡巴斯汀敏感。通过在Ultrogel AcA 34上进行凝胶过滤在天然条件下以及在光亲和标记后通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳在变性条件下测定可溶性神经降压素受体的分子量约为100,000。
