Koeppe J K, Kovalick G E, Prestwich G D
J Biol Chem. 1984 Mar 10;259(5):3219-23.
A tritium-labeled diazocarbonyl juvenile hormone (JH) analog, (10-[10,11-3H]epoxyfarnesyl diazoacetate, [3H]EFDA), covalently bound to proteins in both hemolymph and ovarian extracts when reaction mixtures were irradiated with UV light. The addition of various concentrations of unlabeled JH III selectively inhibited [3H]EFDA photoattachment to proteins. Using the Scatchard method of analysis, [3H]EFDA bound specifically and with relatively high affinity (KD = 1.5 X 10(-6) M) to a macromolecule in each extract, although nonspecific binding to other molecules was also present (20-50%). To determine if [3H]EFDA bound at the JH III-binding site on the binding proteins, radioactive [3H]JH III or [3H]EFDA was complexed with proteins in the presence of various concentrations of either unlabeled JH III or JH I under equilibrium conditions. The results demonstrated that the natural hormone, JH III, displaced both bound labeled ligands 4.1 +/- 0.5 times better than the homolog JH I. Thus, the photoaffinity label [3H]EFDA bound at the same site on the protein as [3H] JH III. Fluorescent autoradiography of [3H]EFDA-labeled proteins separated by sodium dodecyl sulfate electrophoresis revealed that several proteins in both hemolymph and ovarian extracts bound [3H]EFDA. To determine the specificity of binding, extracts were irradiated with UV light in the presence of unlabeled JH III and [3H]EFDA. The results demonstrated that JH III prevented photoattachment of [3H]EFDA to a major protein in each extract. The molecular weight of these proteins was estimated at approximately 200,000 for both the hemolymph protein and the ovarian protein.
当用紫外光照射反应混合物时，一种氚标记的重氮羰基保幼激素（JH）类似物，（10-[10,11-³H]环氧法呢基重氮乙酸酯，[³H]EFDA），与血淋巴和卵巢提取物中的蛋白质共价结合。添加不同浓度的未标记JH III可选择性抑制[³H]EFDA与蛋白质的光附着。使用Scatchard分析方法，[³H]EFDA特异性地且以相对高的亲和力（KD = 1.5×10⁻⁶ M）与每种提取物中的一种大分子结合，尽管也存在与其他分子的非特异性结合（20 - 50%）。为了确定[³H]EFDA是否结合在结合蛋白上的JH III结合位点，在平衡条件下，放射性[³H]JH III或[³H]EFDA与蛋白质在不同浓度的未标记JH III或JH I存在下复合。结果表明，天然激素JH III置换结合的标记配体的能力比同系物JH I强4.1±0.5倍。因此，光亲和标记[³H]EFDA与[³H]JH III结合在蛋白质的同一位点。通过十二烷基硫酸钠电泳分离的[³H]EFDA标记蛋白质的荧光放射自显影显示，血淋巴和卵巢提取物中的几种蛋白质结合了[³H]EFDA。为了确定结合的特异性，提取物在未标记JH III和[³H]EFDA存在下用紫外光照射。结果表明，JH III阻止了[³H]EFDA与每种提取物中的一种主要蛋白质的光附着。这些蛋白质的分子量估计血淋巴蛋白和卵巢蛋白均约为200,000。
