FLUORESCENCE PROBE MEASUREMENT OF INTRALYSOSOMAL PH IN LIVING CELLS AND PERTURBATION OF PH BY VARIOUS AGENTS
FLUORESCENCE PROBE MEASUREMENT OF INTRALYSOSOMAL PH IN LIVING CELLS AND PERTURBATION OF PH BY VARIOUS AGENTS
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DOI：
10.1073/pnas.75.7.3327
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发表时间：
1978-01-01
影响因子：
11.1
通讯作者：
POOLE, B
中科院分区：
文献类型：
--
作者：
OHKUMA, S;POOLE, B
描述了一种用于测量活细胞 [小鼠腹膜巨噬细胞] 中溶酶体内 pH 的定量方法。异硫氰酸荧光素标记的葡聚糖 (FD) 被内吞并在溶酶体中积聚，并在溶酶体中保持不变而没有明显降解。该化合物的荧光光谱在4-7范围内随pH变化而变化，并且不受FD浓度、离子强度或蛋白质浓度的严重影响。盖玻片上的活细胞安装在荧光分光光度计池中，并且可以用各种介质灌注。巨噬细胞溶酶体内的正常 pH 值似乎为 4.7-4.8，但它可能会短暂降至 4.5。细胞暴露于各种弱碱和酸性 K 离子载体会导致 pH 值升高。 pH 值的变化比弱碱在溶酶体内的积累要快得多。糖酵解抑制剂（2-脱氧葡萄糖）和氧化磷酸化抑制剂（氰化物或叠氮化物）一起添加，而不是单独添加，会导致溶酶体内 pH 值升高。这些结果为溶酶体膜中存在活性质子积累机制提供了证据，并支持了溶酶体通过质子捕获弱碱积累的理论。
A quantitative method is described for the measurement of intralysosomal pH in living cells [mouse peritoneal macrophages]. Fluorescein isothiocyanate-labeled dextran (FD) is endocytized and accumulates in lysosomes where it remains without apparent degradation. The fluorescence spectrum of this compound changes with pH in the range 4-7 and is not seriously affected by FD concentration, ionic strength or protein concentration. Living cells on coverslips are mounted in a spectrofluorometer cell and can be perfused with various media. The normal pH inside macrophage lysosomes seems to be 4.7-4.8, although it can drop transiently as low as 4.5. Exposure of the cells to various weak bases and to acidic K ionophores causes the pH to increase. The changes in pH are much more rapid than is the intralysosomal accumulation of the weak bases. Inhibitors of glycolysis (2-deoxyglucose) and of oxidative phosphorylation (cyanide or azide) added together, but not separately, cause the intralysosomal pH to increase. These results provide evidence for the existence of an active proton accumulation mechanism in the lysosomal membrane and support the theory of lysosomal accumulation of weak bases by proton trapping.