Benchimol M, De Souza W, Vanderheyden N, Zhong L, Lu H G, Moreno S N, Docampo R
Universidade Santa Ursula, Rio de Janeiro, RJ 22231-010, Brazil.
Biochem J. 1998 Jun 15;332 ( Pt 3)(Pt 3):695-702. doi: 10.1042/bj3320695.
Acid-loaded Trypanosoma cruzi amastigotes and trypomastigotes regained normal cytoplasmic pH (pHi), as measured in cells loaded with 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF), by a process that was sensitive to bafilomycin A1 at concentrations comparable to those that inhibited vacuolar (V) H+-ATPases from different sources. Steady-state pHi was also decreased by similar concentrations of bafilomycin A1 in a concentration-dependent manner. The efflux of H+ equivalents from amastigotes and trypomastigotes was measured by following changes in the fluorescence of extracellular BCECF. Basal H+ extrusion in the presence of glucose was 15.4+/-2.8 (S.D.) nmol of H+/min per 10(8) amastigotes and 6. 37+/-0.8 nmol of H+/min per 10(8) trypomastigotes. Bafilomycin A1 treatment significantly decreased the efflux of H+ equivalents by amastigotes (8.9+/-2.2 nmol of H+/min per 10(8) cells), but not by trypomastigotes (5.1+/-1.7 nmol of H+/min per 10(8) cells). The localization of the V-H+-ATPase of T. cruzi was investigated by immunocytochemistry. Confocal and electron microscopy indicated that, in addition to being located in cytoplasmic vacuoles, the V-H+-ATPase of different stages of T. cruzi is also located in the plasma membrane. However, no labelling was detected in the plasma membrane lining the flagellar pocket of the different developmental stages. Surface localization of the V-H+-ATPase was confirmed by experiments involving the biotinylation of cell surface proteins and immunoprecipitation with antibodies against the V-H+-ATPase. Taken together, the results are consistent with the presence of a functional V-H+-ATPase in the plasma membrane of amastigotes and with an important role for intracellular acidic compartments in the maintenance of pHi in different stages of T. cruzi.
用2',7'-双-(2-羧乙基)-5(6)-羧基荧光素（BCECF）加载细胞后测量发现，酸性加载的克氏锥虫无鞭毛体和上鞭毛体恢复了正常的细胞质pH值（pHi），该过程对巴弗洛霉素A1敏感，其浓度与抑制来自不同来源的液泡（V）H⁺-ATP酶的浓度相当。相似浓度的巴弗洛霉素A1也以浓度依赖的方式降低了稳态pHi。通过跟踪细胞外BCECF荧光的变化来测量无鞭毛体和上鞭毛体中H⁺当量的外流。在有葡萄糖存在的情况下，基础H⁺外排量为每10⁸个无鞭毛体15.4±2.8（标准差）nmol H⁺/分钟，每10⁸个上鞭毛体6.37±0.8 nmol H⁺/分钟。巴弗洛霉素A1处理显著降低了无鞭毛体的H⁺当量外流（每10⁸个细胞8.9±2.2 nmol H⁺/分钟），但对上鞭毛体没有影响（每10⁸个细胞5.1±1.7 nmol H⁺/分钟）。通过免疫细胞化学研究了克氏锥虫V-H⁺-ATP酶的定位。共聚焦显微镜和电子显微镜表明，除了位于细胞质液泡中外，克氏锥虫不同阶段的V-H⁺-ATP酶也位于质膜中。然而，在不同发育阶段鞭毛袋内衬的质膜中未检测到标记。通过涉及细胞表面蛋白生物素化和用抗V-H⁺-ATP酶抗体进行免疫沉淀的实验证实了V-H⁺-ATP酶在表面的定位。综上所述，这些结果与无鞭毛体质膜中存在功能性V-H⁺-ATP酶以及细胞内酸性区室在维持克氏锥虫不同阶段pHi中的重要作用一致。
