Calahorra M, Martínez G A, Hernández-Cruz A, Peña A
Department of Microbiology, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, México DF.
Yeast. 1998 Apr 30;14(6):501-15. doi: 10.1002/(SICI)1097-0061(19980430)14:6<501::AID-YEA249>3.0.CO;2-6.
The effects of monovalent cations on the internal pH of yeast were studied. Our former procedure was modified, inducing maximal alkalinization of the cells with 100 mM-NH4OH instead of Tris base. The pH values were lower than reported before (Peña et al., J. Baceteriol. 1995 177, 1017-1022). With glucose as substrate, the internal cytoplasmic pH reached higher values when incubating at an external pH of 6.0, as compared to pH 4.0. Monovalent cations added approximately 5 min after glucose produced a further increase in the internal pH, which was higher at a previous incubation pH of 4.0 than that observed at pH 6.0. The selectivity of the changes followed a similar order to that of the transport system for monovalent cations. When incubating cells with glucose for more than 30 min, the initial changes of the internal pH appeared to be regulated by the cell. However, under the fluorescence microscope, it was observed that pyranine, which was confined to the cytoplasm during the first 15 min, was progressively concentrated in the vacuole. By studying the fluorescence changes of cells electroporated and then incubated with glucose or glucose plus potassium, we could follow the internal pH of this organelle, obtaining values within the range reported by other authors. Also, in cells preincubated with glucose for 60 min, and electroporated afterwards, the fluorescence of pyranine, which only entered the cytoplasm, allowed us to measure the pH of this compartment, showing that it was more alkaline than the vacuole. Moreover, the cytoplasmic pH increased upon addition of glucose or potassium. The vacuolar pH, on the other hand, increased upon addition of potassium after glucose, but decreased upon addition of glucose. In addition, incubation of the cells with glucose with or without pyranine produced vesiculation of the vacuole.
研究了单价阳离子对酵母细胞内pH值的影响。我们改进了之前的实验方法，用100 mM - NH₄OH而非Tris碱诱导细胞达到最大碱化程度。得到的pH值比之前报道的要低（Peña等人，《细菌学杂志》1995年，第177卷，第1017 - 1022页）。以葡萄糖为底物时，与外部pH值为4.0相比，在外部pH值为6.0下孵育时，细胞质内pH值会达到更高的值。在加入葡萄糖约5分钟后添加单价阳离子会使细胞内pH值进一步升高，在之前孵育pH值为4.0时升高幅度比在pH值为6.0时更高。这些变化的选择性遵循与单价阳离子转运系统相似的顺序。当用葡萄糖孵育细胞超过30分钟时，细胞内pH值的初始变化似乎受细胞调节。然而，在荧光显微镜下观察到，在前15分钟局限于细胞质中的吡喃荧光素逐渐集中到液泡中。通过研究电穿孔后再用葡萄糖或葡萄糖加钾孵育的细胞的荧光变化，我们可以追踪该细胞器的细胞内pH值，得到的数值在其他作者报道的范围内。同样，在预先用葡萄糖孵育60分钟后再进行电穿孔的细胞中，仅进入细胞质的吡喃荧光素的荧光使我们能够测量该隔室的pH值，结果表明其比液泡的pH值更偏碱性。此外，添加葡萄糖或钾后细胞质pH值升高。另一方面，在葡萄糖之后添加钾时液泡pH值升高，但添加葡萄糖时液泡pH值降低。另外，用含或不含吡喃荧光素的葡萄糖孵育细胞会导致液泡形成囊泡。
