INTRACELLULAR PH MEASUREMENTS IN EHRLICH ASCITES TUMOR-CELLS UTILIZING SPECTROSCOPIC PROBES GENERATED INSITU
INTRACELLULAR PH MEASUREMENTS IN EHRLICH ASCITES TUMOR-CELLS UTILIZING SPECTROSCOPIC PROBES GENERATED INSITU
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DOI：
10.1021/bi00578a012
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发表时间：
1979-01-01
期刊：
影响因子：
2.9
通讯作者：
RACKER, E
中科院分区：
文献类型：
--
作者：
THOMAS, JA;BUCHSBAUM, RN;RACKER, E
不带电荷的无色分子荧光素二乙酸酯在中性pH下扩散到[小鼠]埃利希腹水肿瘤细胞中，其中细胞内酯酶释放发色团荧光素。带负电荷的染料被细胞保留，允许从pH依赖性吸收光谱的形状估计细胞内pH。6-羧基荧光素的二乙酸酯衍生物可以类似地使用，并且具有从细胞中渗漏出的速率较慢的额外优点，但需要在pH 6.2下孵育以促进初始进入细胞。通过离心去除外部染料后，80-92%的剩余染料对外部pH变化无反应。细胞内荧光素光谱的校准是通过在K+缓冲液中用尼日利亚菌素平衡内部和外部pH来获得的。在各种代谢条件下，用这种方法测量细胞内pH的结果与通过测量弱酸性5，5-二甲基[2- 14 C]恶唑烷-2，4-二酮的分配比获得的结果一致。除了细胞内pH值的准确估计，该方法允许细胞内pH值变化的动力学小至0.01以下。细胞内荧光素报告细胞质和线粒体室中发生的pH变化，而6-羧基荧光素仅报告细胞质室。在同等浓度下，尼日利亚菌素比缬氨霉素加质子载体1799在消散质膜pH梯度方面更有效。两者在较低浓度下均有效消散线粒体pH梯度。向埃利希腹水细胞中加入葡萄糖导致细胞质的短暂酸化，与细胞内乳酸水平密切相关。瞬时酸化可以通过糖酵解的初始快速速率超过乳酸输出速率来解释。
The uncharged, colorless molecule fluorescein diacetate diffuses into [mouse] Ehrlich ascites tumor cells at neutral pH, where intracellular esterases release the chromophore fluorescein. The negatively charged dye is retained by the cell, permitting the intracellular pH to be estimated from the shape of the pH-dependent absorption spectrum. The diacetate derivative of 6-carboxyfluorescein may be used similarly and has the additional advantage of a slower rate of leakage out of the cell but requires incubation at pH 6.2 to facilitate initial entry into the cell. After removal of external dye by centrifugation, 80-92% of the remaining dye is unresponsive to external pH changes. Calibration of the intracellular fluorescein spectra is obtained by equilibration of the internal and external pH with nigericin in K+ buffers. Results of intracellular pH measurements by this method agree with those obtained by measuring the distribution ratio of the weak acid 5,5-dimethyl[2-14C]oxazolidine-2,4-dione, under various metabolic conditions. Besides the accurate estimation of intracellular pH, the method permits the kinetics of intracellular pH changes as small as 0.01 to be followed. Intracellular fluorescein reports pH changes occurring in both the cytoplasmic and the mitochondrial compartments, whereas 6-carboxyfluorescein reports only the cytoplasmic compartment. At equivalent concentrations, nigericin is more effective than valinomycin plus the protonophore 1799 in dissipating plasmalemma pH gradients. Both are effective at lower concentrations in dissipating mitochondrial pH gradients. Addition of glucose to Ehrlich ascites cells results in a transient acidification of the cytoplasm in close correspondence to the intracellular lactate levels. The transient acidification can be explained by the initial rapid rate of glycolysis exceeding the rate of lactate export.