ACIDIFICATION OF MACROPHAGE AND FIBROBLAST ENDOCYTIC VESICLES INVITRO
ACIDIFICATION OF MACROPHAGE AND FIBROBLAST ENDOCYTIC VESICLES INVITRO
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DOI：
10.1073/pnas.80.11.3334
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
MELLMAN, I
中科院分区：
文献类型：
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作者：
GALLOWAY, CJ;DEAN, GE;MELLMAN, I
使用pH依赖性荧光染料荧光素-葡聚糖（FD）研究从培养的[小鼠]巨噬细胞和[仓鼠]成纤维细胞分离的前溶酶体空泡（内体）和溶酶体的酸化。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通过胞饮内化。成纤维细胞也暴露于20 ℃的FD。C，在该温度下内体-溶酶体融合被抑制。将细胞均质化，并通过在Percoll密度梯度中离心分离标记的细胞器。ATP的加入迅速降低了内体和溶酶体的内部pH值，如荧光强度降低所示。pH梯度被H+离子载体和氯化铵消散。酸化不受线粒体F1，F0-ATP酶或Na+，K+-ATP酶抑制剂的影响，并不需要渗透阴离子，Na+，或K+。在测试的抑制剂中，仅N-乙基马来酰亚胺防止两个隔室的ATP依赖性酸化。这些研究结果提供了直接支持的存在一个酸性的前溶酶体室，可以通过相同类型的H+泵被认为是在溶酶体和分泌颗粒酸化。
The pH-dependent fluorochrome fluorescein-dextran (FD) was used to study the acidification of prelysosomal vacuoles (endosomes) and lysosomes isolated from cultured [mouse] macrophages and [hamster] 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.degree. 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+-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.