Deng Y, Bennink J R, Kang H C, Haugland R P, Yewdell J W
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, NIH, Bethesda, MD 20892, USA.
J Histochem Cytochem. 1995 Sep;43(9):907-15. doi: 10.1177/43.9.7543914.
The fungal metabolite brefeldin A (BFA) interferes with vesicular trafficking in most animal cells. To gain insight into the mechanism of BFA action, we esterified it to the fluorophore, boron dipyromethene difluoride (BODIPY). BODIPY-BEA localized predominantly in the endoplasmic reticulum (ER) and Golgi complex of viable cells and was extracted by detergent treatment, suggesting it interacts primarily with lipid bilayers. The localization of the conjugate is conferred by BFA, since free BODIPY or BODIPY esterified to cyclopentanol did not specifically localize to internal membranes. BODIPY-BFA exhibited a similar biological activity to BFA, but only when used at higher concentrations and after a delay. HPLC analysis revealed that over this period, cells converted BODIPY-BFA to species co-eluting with free BODIPY and BFA. Therefore, BODIPY-BFA is probably inactive until BFA is released by cellular esterases. The specific localization of BODIPY-BFA to the ER and Golgi complex suggests that BFA might exert its effects on vesicular trafficking by perturbing the lipid bilayer of its target organelles. Because BODIPY-BFA intensely stains the ER at concentrations that have no discernible effects on intracellular transport or other cellular functions, it should be useful for visualizing the ER in living cells.
真菌代谢产物布雷菲德菌素A（BFA）会干扰大多数动物细胞中的囊泡运输。为深入了解BFA的作用机制，我们将其与荧光团二氟化硼二吡咯亚甲基（BODIPY）酯化。BODIPY-BFA主要定位于活细胞的内质网（ER）和高尔基体复合体，经去污剂处理后可被提取，这表明它主要与脂质双层相互作用。共轭物的定位是由BFA赋予的，因为游离的BODIPY或与环戊醇酯化的BODIPY不会特异性定位于内膜。BODIPY-BFA表现出与BFA相似的生物活性，但仅在较高浓度下使用且有延迟时才会如此。高效液相色谱分析表明，在此期间，细胞将BODIPY-BFA转化为与游离BODIPY和BFA共洗脱的物质。因此，在细胞酯酶释放出BFA之前，BODIPY-BFA可能是无活性的。BODIPY-BFA对ER和高尔基体复合体的特异性定位表明，BFA可能通过扰乱其靶细胞器的脂质双层对囊泡运输发挥作用。由于BODIPY-BFA在对细胞内运输或其他细胞功能无明显影响的浓度下就能强烈染色ER，它应有助于在活细胞中可视化ER。
