Wada S, Matsunaga S, Saito S, Fusetani N, Watabe S
Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan.
J Biochem. 1998 May;123(5):946-52. doi: 10.1093/oxfordjournals.jbchem.a022029.
An actin-depolymerizing marine natural product, mycalolide B, and a related compound, kabiramide D, were labeled with biocytin, a biotin derivative, and used to specify target molecules in cultured rat 3Y1 fibroblasts. Mycalolide B exhibited the ability to bind to various intracellular proteins, probably through the Michael addition of a sulfhydryl group to C5 of mycalolide B. However, no intracellular proteins other than actin apparently reacted with biocytinylated kabiramide D, demonstrating that the binding of kabiramide D to actin was highly specific. Cells treated with biocytinylated kabiramide D followed by staining with fluorescein isothiocyanate-conjugated avidin showed that biocytinylated kabiramide D bound to stress fibers composed of F-actin, although the staining intensity was weaker than the fluorescent phalloidin staining. The assay for the binding of kabiramide D to actin, which had previously been treated with other actin-depolymerizing agents, showed that the actin-binding site for kabiramide D was the same as that for bistheonellide A, but not those for latrunculin A and cytochalasin D.
一种肌动蛋白解聚海洋天然产物麦考内酯B和一种相关化合物卡比拉酰胺D，用生物素（一种生物素衍生物）进行标记，并用于确定培养的大鼠3Y1成纤维细胞中的靶分子。麦考内酯B表现出与多种细胞内蛋白质结合的能力，可能是通过巯基对麦考内酯B的C5进行迈克尔加成。然而，除肌动蛋白外，显然没有其他细胞内蛋白质与生物素化的卡比拉酰胺D发生反应，这表明卡比拉酰胺D与肌动蛋白的结合具有高度特异性。用生物素化的卡比拉酰胺D处理细胞，然后用异硫氰酸荧光素偶联抗生物素蛋白染色，结果显示生物素化的卡比拉酰胺D与由F-肌动蛋白组成的应力纤维结合，尽管染色强度比荧光鬼笔环肽染色弱。对卡比拉酰胺D与先前用其他肌动蛋白解聚剂处理过的肌动蛋白结合的测定表明，卡比拉酰胺D的肌动蛋白结合位点与双硫内酯A相同，但与拉特鲁菌素A和细胞松弛素D的不同。
