Kovalick G E, Koeppe J K
Mol Cell Endocrinol. 1983 Aug;31(2-3):271-86. doi: 10.1016/0303-7207(83)90154-5.
We modified a binding assay using polyethylene glycol (PEG) to precipitate bound hormone. Optimum precipitation occurred when reaction mixtures were incubated with 10-40% PEG and 1.25-2.5 mg/ml gamma-globulins for 2-90 min at 4 or 23 degrees C. Results from this assay and from the dextran-coated charcoal assay were similar. Addition of phenylmethylsulfonyl fluoride eliminated nonspecific esterase activity in extracts. JH III-binding macromolecules were identified in hemolymph and ovaries of Leucophaea maderae. These molecules were pronase- and heat-sensitive and saturable. Using Scatchard analysis an average KD of 2.04 (+/- 0.32) X 10(-8) M and 1.91 (+/- 0.80) X 10(-8) M was calculated for hemolymph and ovarian binding proteins. JH III had the highest affinity for binding sites, followed by JH I and JH 0. Various extraction procedures caused changes in JH affinity for both binding proteins. At high concentrations the (+) isomer and mixed isomer preparations of methoprene and hydroprene competed for binding sites. Binding proteins had no affinity for the (-) isomer or for the JH III acid.
我们改进了一种结合测定法，使用聚乙二醇（PEG）沉淀结合的激素。当反应混合物在4或23摄氏度下与10 - 40%的PEG和1.25 - 2.5毫克/毫升的γ球蛋白孵育2 - 90分钟时，会出现最佳沉淀。该测定法和葡聚糖包被活性炭测定法的结果相似。加入苯甲基磺酰氟可消除提取物中的非特异性酯酶活性。在马德拉蜚蠊的血淋巴和卵巢中鉴定出了JH III结合大分子。这些分子对链霉蛋白酶和热敏感且具有饱和性。使用Scatchard分析，计算出血淋巴和卵巢结合蛋白的平均解离常数（KD）分别为2.04（±0.32）×10⁻⁸ M和1.91（±0.80）×10⁻⁸ M。JH III对结合位点的亲和力最高，其次是JH I和JH 0。各种提取程序导致JH对两种结合蛋白的亲和力发生变化。在高浓度下，烯虫酯和烯虫炔酯的（+）异构体和混合异构体制剂会竞争结合位点。结合蛋白对（-）异构体或JH III酸没有亲和力。
