Galloway C J, Dean G E, Marsh M, Rudnick G, Mellman I
Proc Natl Acad Sci U S A. 1983 Jun;80(11):3334-8. doi: 10.1073/pnas.80.11.3334.
We have used the pH-dependent fluorochrome fluorescein-dextran (FD) to study the acidification of prelysosomal vacuoles (endosomes) and lysosomes isolated from cultured macrophages and fibroblasts. FD was internalized by pinocytosis under conditions that allowed its selective localization in endosomes (1- to 5-min pulse) or in lysosomes (5-min pulse, 30-min chase). Fibroblasts were also exposed to FD at 20 degrees C, at which temperature endosome-lysosome fusion is inhibited. Cells were homogenized and labeled organelles were separated by centrifugation in Percoll density gradients. The addition of ATP rapidly decreased the internal pH of both endosomes and lysosomes, as indicated by a decrease in fluorescence intensity. The pH gradient was dissipated by H+ ionophores and ammonium chloride. Acidification was not affected by inhibitors of the mitochondrial F1, F0-ATPase or the Na+, K, K+-ATPase and did not require permeant anions, Na+, or K+. Of the inhibitors tested, only N-ethylmaleimide prevented the ATP-dependent acidification of both compartments. These findings provide direct support for the existence of an acidic prelysosomal compartment that may be acidified via the same type of H+ pump believed to operate in lysosomes and secretory granules.
我们使用了pH依赖性荧光染料荧光素-葡聚糖（FD）来研究从培养的巨噬细胞和成纤维细胞中分离出的前溶酶体空泡（内体）和溶酶体的酸化情况。在允许其选择性定位于内体（1至5分钟脉冲）或溶酶体（5分钟脉冲，30分钟追踪）的条件下，FD通过胞饮作用被内化。成纤维细胞也在20℃下暴露于FD，在该温度下内体-溶酶体融合受到抑制。细胞被匀浆，标记的细胞器通过在Percoll密度梯度中离心分离。ATP的添加迅速降低了内体和溶酶体的内部pH，荧光强度降低表明了这一点。H⁺离子载体和氯化铵使pH梯度消散。酸化不受线粒体F1、F0-ATP酶或Na⁺、K⁺、K⁺-ATP酶抑制剂的影响，并且不需要渗透性阴离子、Na⁺或K⁺。在测试的抑制剂中，只有N-乙基马来酰亚胺阻止了两个区室的ATP依赖性酸化。这些发现为存在一个酸性前溶酶体区室提供了直接支持，该酸性前溶酶体区室可能通过与据信在溶酶体和分泌颗粒中起作用的相同类型的H⁺泵酸化。
