McKernan R M, Gillard N P, Quirk K, Kneen C O, Stevenson G I, Swain C J, Ragan C I
Department of Biochemistry, Merck Sharp and Dohme Research Laboratories, Harlow, Essex, United Kingdom.
J Biol Chem. 1990 Aug 15;265(23):13572-7.
A 5-hydroxytryptamine 5-HT3 receptor binding site has been purified from deoxycholate-solubilized NCB20 cell membranes. Purification (1,700-fold) was achieved in one step by affinity chromatography with L-685,603 immobilized on agarose. The 5-HT3 selective antagonist [3H]Q ICS 205-930 labeled a single population of receptors in the affinity-purified preparation with a Bmax of 3.1 +/- 0.9 nmol/mg protein and Kd of 0.40 +/- 0.05 nM (mean +/- S.E., n = 3). The rank order of potency for a series of competing compounds confirmed that [3H]Q ICS 205,930 was labeling a 5-HT3 receptor in the purified preparation, and the inhibition constants for all antagonists were unchanged after purification. The purified 5-HT3 binding site eluted from a Sepharose 6B gel filtration column in a similar manner to the crude solubilized preparation (Stokes radius of 4.9 nm, apparent molecular size 250,000). Polyacrylamide gel electrophoresis of the affinity-purified receptor showed two broad bands by silver staining, migrating with apparent molecular masses of 54,000 and 38,000. Gel filtration of the affinity purified material yielded a single peak labeled by [3H]Q ICS 205-930 with an apparent molecular size of 250,000, which was also composed of two bands of 54,000 and 38,000, consistent with these being the constituents of the 5-HT3 receptor.
已从脱氧胆酸盐增溶的NCB20细胞膜中纯化出5-羟色胺5-HT3受体结合位点。通过用固定在琼脂糖上的L-685,603进行亲和层析一步实现了纯化（1700倍）。5-HT3选择性拮抗剂[3H]Q ICS 205-930在亲和纯化制剂中标记了单一受体群体，Bmax为3.1±0.9 nmol/mg蛋白，Kd为0.40±0.05 nM（平均值±标准误，n = 3）。一系列竞争化合物的效价顺序证实[3H]Q ICS 205,930在纯化制剂中标记的是5-HT3受体，并且所有拮抗剂的抑制常数在纯化后未发生变化。从琼脂糖6B凝胶过滤柱上洗脱的纯化5-HT3结合位点，其洗脱方式与粗增溶制剂相似（斯托克斯半径为4.9 nm，表观分子大小为250,000）。亲和纯化受体的聚丙烯酰胺凝胶电泳经银染显示出两条宽带，表观分子量分别为54,000和38,000。亲和纯化材料的凝胶过滤产生了一个由[3H]Q ICS 205-930标记的单峰，表观分子大小为250,000，它也由54,000和38,000的两条带组成，这与它们是5-HT3受体的组成成分一致。
