Lehmann M, Koolman J
Physiologisch-Chemisches Institut, Philipps-Universität, Marburg, F.R.G.
Mol Cell Endocrinol. 1988 Jun;57(3):239-49. doi: 10.1016/0303-7207(88)90080-9.
A macromolecule with high affinity for the ecdysteroid analogue ponasterone A was isolated from nuclei of larvae of the blowfly Calliphora vicina. The ecdysteroid-binding molecule revealed characteristics of the moulting hormone receptor. It was sensitive towards protease but not towards nucleases. The nuclear protein had a limited binding capacity (0.2 pmol ponasterone A/mg protein), showed hormone analogue specificity and high affinity for ecdysteroids. Enzyme activities were present in the nuclear extract that metabolized ecdysteroids and thereby interfered with the binding assay. After their removal by DEAE-cellulose chromatography the ecdysteroid receptor preparation was stable at 20 degrees C for hours. This allowed a reliable determination of dissociation constants at equilibrium conditions. The hormone receptor complex had a KD of 1 nM, 30 nM, and 2000 nM with ponasterone A, 20-hydroxyecdysone, and ecdysone, respectively. The apparent molecular mass of the ecdysteroid receptor was 105,000 as determined by gel filtration.
从丽蝇（Calliphora vicina）幼虫的细胞核中分离出一种对蜕皮甾类类似物ponasterone A具有高亲和力的大分子。这种蜕皮甾类结合分子显示出蜕皮激素受体的特征。它对蛋白酶敏感，但对核酸酶不敏感。该核蛋白的结合能力有限（0.2 pmol ponasterone A/mg蛋白），对激素类似物具有特异性，且对蜕皮甾类具有高亲和力。核提取物中存在代谢蜕皮甾类的酶活性，从而干扰了结合测定。通过DEAE-纤维素色谱法去除这些酶活性后，蜕皮甾类受体制剂在20℃下可稳定数小时。这使得在平衡条件下能够可靠地测定解离常数。激素受体复合物与ponasterone A、20-羟基蜕皮酮和蜕皮酮的解离常数分别为1 nM、30 nM和2000 nM。通过凝胶过滤测定，蜕皮甾类受体的表观分子量为105,000。
