Liu J J, Kelly J, Casley D J, Johnston C I, Nayler W G
Department of Medicine, University of Melbourne, Austin Hospital, Heidelberg, Victoria, Australia.
Biochem Biophys Res Commun. 1990 Jan 15;166(1):299-307. doi: 10.1016/0006-291x(90)91945-o.
Adult rat cardiac endothelin-1 (ET-1) receptors were solubilized with 0.5% digitonin and then characterized. The receptors retained binding activity after solubilization. Binding was saturable (KD of 0.065 +/- 0.004 nM, Bmax of 94.6 +/- 4.5 fmol/mg protein; Hill coefficient of 0.987 +/- 0.017 n = 6) and pH dependent, with the binding increasing as the pH was decreased from 10 to 4, but decreasing dramatically as pH dropped to 2. Specifically bound [125I]-ET-1 was not dissociated by 2 x 10(-7) M unlabelled ET-1, but was dissociated by pH 10 and 2. Returning the pH to 7.4 restored the binding activity of the receptors. Unlabelled ET-1 (10(-12) - 10(-7) M) and sarafotoxin S6b(10(-12) - 10(-7) M) competed with [125I]-ET-1 for binding to the receptors.
用0.5%洋地黄皂苷溶解成年大鼠心脏内皮素-1（ET-1）受体，然后对其进行特性鉴定。受体在溶解后仍保留结合活性。结合具有饱和性（解离常数KD为0.065±0.004 nM，最大结合容量Bmax为94.6±4.5 fmol/mg蛋白质；希尔系数为0.987±0.017，n = 6）且依赖于pH值，随着pH值从10降至4，结合增加，但当pH值降至2时则急剧下降。特异性结合的[125I]-ET-1不会被2×10⁻⁷ M未标记的ET-1解离，但会被pH值为10和2的溶液解离。将pH值恢复到7.4可恢复受体的结合活性。未标记的ET-1（10⁻¹² - 10⁻⁷ M）和萨拉索毒素S6b（10⁻¹² - 10⁻⁷ M）与[125I]-ET-1竞争结合受体。
