Gásková D, Brodská B, Herman P, Vecer J, Malínský J, Sigler K, Benada O, Plásek J
Institute of Physics, Charles University, Prague, Czech Republic.
Yeast. 1998 Sep 30;14(13):1189-97. doi: 10.1002/(sici)1097-0061(19980930)14:13<1189::aid-yea320>3.3.co;2-b.
Membrane-potential-dependent accumulation of diS-C3(3) in intact yeast cells in suspension is accompanied by a red shift of the maximum of its fluorescence emission spectrum, lambda max, caused by a readily reversible probe binding to cell constituents. Membrane depolarization by external KCl (with or without valinomycin) or by ionophores causes a fast and reproducible blue shift. As the potential-reporting parameter, the lambda max shift is less affected by probe binding to cuvette walls and possible photobleaching than, for example, fluorescence intensity. The magnitude of the potential-dependent red lambda max shift depends on relative cell-to-probe concentration ratio, a maximum shift (572-->582 nm) being found in very thick suspensions and in cell lysates. The potential therefore has to be assessed at reasonably low cell (< or = 5 x 10(6) cells/ml) and probe (10(-7)M) concentrations at which a clearly defined relationship exists between the lambda max shift and the potential-dependent accumulation of the dye in the cells. The redistribution of the probe between the medium and yeast protoplasts takes about 5 min, but in intact cells it takes 10-30 min because the cell wall acts as a barrier, hampering probe penetration into the cells. The barrier properties of the cell wall correlate with its thickness: cells grown in 0.2% glucose (cell wall thickness 0.175 +/- 0.015 micron, n = 30) are stained much faster and the lambda max is more red-shifted than in cells grown in 2% glucose (cell wall thickness 0.260 +/- 0.043 micron, n = 44). At a suitable cell and probe concentration and under standard conditions, the lambda max shift of diS-C3(3) fluorescence provides reliable information on even fast changes in membrane potential in Saccharomyces cerevisiae.
悬浮状态下完整酵母细胞中，二硫代-C3(3) 依赖膜电位的积累伴随着其荧光发射光谱最大值（λmax）的红移，这是由于探针与细胞成分的可逆结合所致。外部氯化钾（有或没有缬氨霉素）或离子载体引起的膜去极化会导致快速且可重复的蓝移。作为电位报告参数，与例如荧光强度相比，λmax 位移受探针与比色皿壁结合以及可能的光漂白影响较小。电位依赖性红移 λmax 的大小取决于细胞与探针的相对浓度比，在非常浓的悬浮液和细胞裂解物中发现最大位移（572→582 nm）。因此，必须在合理低的细胞（≤5×10⁶ 个细胞/毫升）和探针（10⁻⁷M）浓度下评估电位，此时 λmax 位移与染料在细胞中的电位依赖性积累之间存在明确的关系。探针在培养基和酵母原生质体之间的重新分布约需5分钟，但在完整细胞中则需10 - 30分钟，因为细胞壁起到屏障作用，阻碍探针进入细胞。细胞壁的屏障特性与其厚度相关：在0.2%葡萄糖中生长的细胞（细胞壁厚度0.175±0.015微米，n = 3）比在2%葡萄糖中生长的细胞（细胞壁厚度0.260±0.043微米，n = 44）染色快得多，且λmax红移更大。在合适的细胞和探针浓度以及标准条件下，二硫代-C3(3) 荧光的λmax 位移为酿酒酵母膜电位的快速变化提供了可靠信息。
