Onda Maki, Inoue Yukari, Kawabata Manami, Mita Tomoyoshi
Department of Environmental Sciences, Faculty of Science, Osaka Women's University, 2-1 Daisen-cho, Sakai, Osaka 590-0035.
J Biochem. 2003 Jul;134(1):121-8. doi: 10.1093/jb/mvg119.
To investigate the susceptibilities of fungal and mammalian cells to amphotericin B (AmB), AmB-loaded lysophosphatidylcholine (LPC)micelles as drug delivery vehicles were incubated at 37 degrees C with phosphatidylcholine vesicles containing different sterols as model systems for fungal and mammalian cells. The binding and kinetics of AmB to sterols in the membranes were judged by UV-visible spectroscopy. In the 91% monomeric form, AmB interacted rapidly with ergosterol and slowly with 7-dehydrocholesterol (7-DHC), while it did not interact with cholesterol. In the 50% monomeric form, AmB formed complexes more rapidly with ergosterol or 7-DHC than in the monomeric form, whereas it did not still interact with cholesterol. The interaction was also characterized by resonance energy transfer between the fluorescent probe trimethylammonium diphenylhexatriene (TMA-DPH) and AmB. In the 91% monomeric form, AmB caused initial fluorescence quenching in bilayer membranes containing any sterol as well as sterol-free bilayer membranes due to the release of AmB and its incorporation within the membranes. However, a second phase of increasing fluorescence was found in the case of ergosterol alone. On the other hand, in the 47% monomeric form, AmB gave a biphasic intensity profile in membranes containing any sterol as well as sterol-free membranes. However, the extent of the second phase of increasing fluorescence intensity was markedly dependent upon sterol composition. Studies using sterol-containing vesicles provide important insights into the role of the aggregation state of AmB in its effects on cells.
为了研究真菌细胞和哺乳动物细胞对两性霉素B（AmB）的敏感性，将负载AmB的溶血磷脂酰胆碱（LPC）胶束作为药物递送载体，在37℃下与含有不同甾醇的磷脂酰胆碱囊泡一起孵育，这些囊泡作为真菌细胞和哺乳动物细胞的模型系统。通过紫外可见光谱法判断AmB与膜中甾醇的结合及动力学。以91%的单体形式存在时，AmB与麦角甾醇快速相互作用，与7-脱氢胆固醇（7-DHC）缓慢相互作用，而与胆固醇不相互作用。以50%的单体形式存在时，AmB与麦角甾醇或7-DHC形成复合物的速度比单体形式更快，而与胆固醇仍不相互作用。这种相互作用还通过荧光探针三甲基铵二苯基己三烯（TMA-DPH）与AmB之间的共振能量转移来表征。以91%的单体形式存在时，由于AmB的释放及其掺入膜中，AmB在含有任何甾醇的双层膜以及无甾醇双层膜中引起初始荧光猝灭。然而，仅在麦角甾醇的情况下发现了荧光增强的第二阶段。另一方面，以47%的单体形式存在时，AmB在含有任何甾醇的膜以及无甾醇膜中呈现双相强度分布。然而，荧光强度增加的第二阶段的程度明显取决于甾醇组成。使用含甾醇囊泡的研究为AmB的聚集状态在其对细胞的作用中的角色提供了重要见解。
