Ohashi M, Murata M, Ohnishi S
Department of Biophysics, Faculty of Science, Kyoto University, Japan.
Eur J Cell Biol. 1992 Oct;59(1):116-26.
A novel fluorescence method to monitor the lysosomal disintegration of low density lipoprotein (LDL) particles in living cells has been developed. The method is based on the fluorescence resonance energy transfer (RET) between two fluorescent molecules incorporated into LDL particles. NBD-cholesterol linoleate (NBD-CL) and octadecyl rhodamine B (R18) were incorporated simultaneously into LDL, as a RET donor and a RET acceptor, respectively. In this preparation of LDL (RET-LDL), efficient RET was observed, and after the disruption of the LDL particle by a Triton X-100 treatment, the relief of the RET was observed. RET-LDL was endocytosed by CHO cells via LDL receptors, and the RET-LDL particles were disintegrated after the uptake. The resultant relief of the RET upon the disintegration of the LDL was monitored by flow cytometry, and the amount of intact LDL in cells was estimated by calculation. The disintegration occurred with an about 25 min lag, and was inhibited by several lysosomal inhibitors. These results indicate that the disintegration was not a nonspecific event, but took place at the level of lysosomes. Since living cells can be analyzed by the present method, when coupled to flow sorting, it would permit the isolation of cells having different properties in the endocytic pathway of LDL.
一种用于监测活细胞中低密度脂蛋白（LDL）颗粒溶酶体解体的新型荧光方法已被开发出来。该方法基于掺入LDL颗粒中的两种荧光分子之间的荧光共振能量转移（RET）。将NBD-胆固醇亚油酸酯（NBD-CL）和十八烷基罗丹明B（R18）分别作为RET供体和RET受体同时掺入LDL中。在这种LDL（RET-LDL）制剂中，观察到了高效的RET，并且在用Triton X-100处理破坏LDL颗粒后，观察到了RET的缓解。RET-LDL通过LDL受体被CHO细胞内吞，并且摄取后RET-LDL颗粒解体。通过流式细胞术监测LDL解体时RET的缓解情况，并通过计算估计细胞中完整LDL的量。解体发生有大约25分钟的延迟，并且受到几种溶酶体抑制剂的抑制。这些结果表明解体不是一个非特异性事件，而是发生在溶酶体水平。由于可以通过本方法分析活细胞，当与流式分选结合时，它将允许分离在LDL内吞途径中具有不同特性的细胞。
