Calahorra M, Ramírez J, Clemente S M, Peña A
Biochim Biophys Acta. 1987 May 29;899(2):229-38. doi: 10.1016/0005-2736(87)90404-4.
Vesicles from yeast plasma membrane were prepared according to Franzusoff and Cirillo [1983) J. Biol. Chem. 258, 3608), with slight modifications. When Mg-ATP was added, this preparation was able to generate a membrane potential, that was sensitive to inhibitors of the yeast H+-ATPase and uncouplers, and could be decreased by the addition of permeant anions, as measured by the fluorescence changes of the dye oxonol V. The addition of ATP could also generate a pH gradient, detectable by the fluorescence changes of the monitor aminochloromethoxyacridine. This gradient was sensitive to inhibitors of ATPase and uncouplers, and could be increased by the addition of permeant anions to the incubation mixture. When the vesicles were loaded with KCl, an increased rate of K+ efflux was produced upon the addition of ATP. Cytochrome oxidase from bovine heart could be reconstituted into the vesicles and was shown to generate a membrane potential difference, negative inside, evidenced by the fluorescence quenching of the cyanide dipropylthiacarbocyanine and the uptake of tetraphenylphosphonium. Besides, in these vesicles, K+ and Rb+, but not Na+ or NH+4 could decrease the quenching of fluorescence and the uptake of tetraphenylphosphonium produced when the electron-donor system was present. In the vesicles in which cytochrome oxidase was incorporated, upon the addition of cytochrome c and ascorbate, the uptake of 86Rb+ could be demonstrated also. This uptake was found to be saturable and inhibited by K+, and to a lesser degree by Na+. The results obtained indicate that these vesicles are reasonably sealed and capable of generating and maintaining a membrane potential. The membrane potential could be used to drive ions across the membrane of the vesicles, indicating the presence and functionality of the monovalent cation carrier. The vesicles, in general terms seem to be suitable for studying transport of ions and metabolites in yeast.
按照Franzusoff和Cirillo [1983]《生物化学杂志》第258卷，第3608页所述方法制备酵母质膜囊泡，并略作修改。添加Mg-ATP时，该制剂能够产生膜电位，该膜电位对酵母H⁺-ATP酶抑制剂和解偶联剂敏感，并且通过添加渗透性阴离子可使其降低，这可通过染料氧化诺V的荧光变化来测量。添加ATP还可产生pH梯度，可通过监测氨基氯甲氧基吖啶的荧光变化来检测。该梯度对ATP酶抑制剂和解偶联剂敏感，并且通过向孵育混合物中添加渗透性阴离子可使其增加。当囊泡装载KCl时，添加ATP会使K⁺外流速率增加。牛心细胞色素氧化酶可重新组装到囊泡中，并显示能产生膜电位差，内部为负，这可通过氰化二丙基硫代羰花青的荧光猝灭和四苯基鏻的摄取来证明。此外，在这些囊泡中，K⁺和Rb⁺，而不是Na⁺或NH₄⁺，可降低存在电子供体系统时产生的荧光猝灭和四苯基鏻的摄取。在掺入细胞色素氧化酶的囊泡中，添加细胞色素c和抗坏血酸后，也可证明⁸⁶Rb⁺的摄取。发现这种摄取是可饱和的，并受到K⁺的抑制，在较小程度上受到Na⁺的抑制。所得结果表明这些囊泡密封性良好，能够产生并维持膜电位。膜电位可用于驱动离子穿过囊泡膜，表明单价阳离子载体的存在及其功能。一般来说，这些囊泡似乎适合用于研究酵母中离子和代谢物的运输。
