RHODAMINE-123 AS A PROBE OF TRANSMEMBRANE POTENTIAL IN ISOLATED RAT-LIVER MITOCHONDRIA - SPECTRAL AND METABOLIC PROPERTIES
RHODAMINE-123 AS A PROBE OF TRANSMEMBRANE POTENTIAL IN ISOLATED RAT-LIVER MITOCHONDRIA - SPECTRAL AND METABOLIC PROPERTIES
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DOI：
10.1016/0005-2728(86)90112-x
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发表时间：
1986-07-23
期刊：
影响因子：
--
通讯作者：
LEMASTERS, JJ
中科院分区：
文献类型：
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作者：
EMAUS, RK;GRUNWALD, R;LEMASTERS, JJ
罗丹明123是一种荧光阳离子染料，用于标记活细胞中的线粒体，在分离的大鼠肝脏线粒体混悬液中研究了罗丹明123的光谱和代谢特性。线粒体通电后，罗丹明123的吸光度和荧光发生红移。高达75%的荧光猝灭也发生了。红移和猝灭随钾扩散电位呈线性变化，但对δ - δ - ph没有响应。这些与能量相关的变化伴随着染料进入基质空间。吸入与排出的浓度比接近4000:1。大量的内化染料被结合。当浓度高于记录这些光谱变化所需的浓度时，罗丹明123抑制adp刺激的线粒体（状态3）呼吸（Ki = 12.mu）。M)和倒置内膜囊泡atp酶活性（Ki = 126 .mu）。M)和部分纯化的f1 - atp酶（Ki =177 μ M）。偶联线粒体较小的Ki是由于能量依赖性罗丹明123摄取到基质中。蛋白质在20 nmol/mg以上(10 .mu。M)，罗丹明123引起能量充沛的线粒体迅速膨胀。即使在使用的最高罗丹明123浓度下，对电子转移反应和耦合的影响也很小或可以忽略不计。-依赖性罗丹明123摄取与罗丹明123结合是活细胞线粒体强烈荧光染色的原因。线粒体atp酶的抑制可能是罗丹明123细胞毒性的原因。在不抑制线粒体功能的浓度下，罗丹明123是一种敏感和特异性的D.psi探针。在分离的线粒体中。
The spectral and metabolic properties of Rhodamine 123, a fluorescent cationic dye used to label mitochondria in living cells, were investigated in suspensions of isolated rat-liver mitochondria. A red shift of Rhodamine 123 absorbance and fluorescence occurred following mitochondrial energization. Fluorescence quenching of as much as 75% also occurred. The red shift and quenching varied linearly with the potassium diffusion potential, but did not respond to .DELTA.pH. These energy-linked changes were accompanied by dye uptake into the matrix space. Concentration ratios, in-to-out, approached 4000:1. A large fraction of internalized dye was bound. At concentrations higher than those needed to record these spectral changes, Rhodamine 123 inhibited ADP-stimulated (State 3) respiration of mitochondria (Ki = 12 .mu.M) and ATPase activity of inverted inner membrane vesicles (Ki = 126 .mu.M) and partially purified F1-ATPase (Ki =177 .mu.M). The smaller Ki for coupled mitochondria was accounted for by energy-dependent Rhodamine 123 uptake into the matrix. Above about 20 nmol/mg protein (10 .mu.M), Rhodamine 123 caused rapid swelling of energized mitochondria. Effects on electron-transfer reactions and coupling were small or negligible even at the highest Rhodamine 123 concentrations employed. .DELTA..psi.-dependent Rhodamine 123 uptake together with Rhodamine 123 binding account for the intense fluorescent staining of mitochondria in living cells. Inhibition of mitochondria ATPase likely accounts for the cytotoxicity of Rhodamine 123. At concentrations which do not inhibit mitochondrial function, Rhodamine 123 is a sensitive and specific probe of D.psi. in isolated mitochondria.