Miyamoto-Lee Y, Shiosaka S, Tohyama M
Department of Neuroanatomy, Osaka University Medical School, Japan.
Peptides. 1991 Sep-Oct;12(5):1001-6. doi: 10.1016/0196-9781(91)90050-y.
Scatchard analysis of saturation curves was performed to compared newborn and adult rat neurotensin receptor using [3H] neurotensin as a tracer. The membrane fraction of newborn rat cerebral cortex has a single population of neurotensin receptor (Kd = 0.13 nM, Bmax = 710 fmol/mg protein), whereas adults have two distinct neurotensin binding sites (high affinity site, Kd1 = 0.13 nM; low affinity site, Kd2 = 20 nM). High affinity neurotensin receptor, solubilized with digitonin, was purified from newborn rat cortex by affinity chromatography. An overall purification of 14,000-fold was achieved. The binding of [3H] neurotensin to the purified receptor is saturable and specific, with a Kd of 0.45 nM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the presence of 2-mercaptoethanol revealed purified material of a single major band of Mr = 55,000.
采用[3H]神经降压素作为示踪剂，对新生大鼠和成年大鼠的神经降压素受体进行饱和曲线的Scatchard分析。新生大鼠大脑皮层的膜部分有单一的神经降压素受体群体（解离常数Kd = 0.13 nM，最大结合容量Bmax = 710 fmol/mg蛋白质），而成年大鼠有两个不同的神经降压素结合位点（高亲和力位点，Kd1 = 0.13 nM；低亲和力位点，Kd2 = 20 nM）。用洋地黄皂苷增溶的高亲和力神经降压素受体，通过亲和层析从新生大鼠皮层中纯化出来。实现了14000倍的总纯化。[3H]神经降压素与纯化受体的结合是可饱和的且具有特异性，解离常数Kd为0.45 nM。在2-巯基乙醇存在下进行的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示，纯化后的物质为一条Mr = 55000的单一主要条带。
