CELLULAR-ENERGY METABOLISM, TRANS-PLASMA AND TRANS-MITOCHONDRIAL MEMBRANE-POTENTIALS, AND PH GRADIENTS IN MOUSE NEURO-BLASTOMA
CELLULAR-ENERGY METABOLISM, TRANS-PLASMA AND TRANS-MITOCHONDRIAL MEMBRANE-POTENTIALS, AND PH GRADIENTS IN MOUSE NEURO-BLASTOMA
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DOI：
10.1073/pnas.76.5.2175
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发表时间：
1979-01-01
影响因子：
11.1
通讯作者：
SILVER, IA
中科院分区：
文献类型：
--
作者：
DEUTSCH, C;ERECINSKA, M;SILVER, IA
本文提出了一种定量评价完整细胞亚细胞区室跨膜电位和pH梯度的方法。将该方法应用于小鼠神经母细胞瘤C-1300克隆NB 41 A3的研究，其中跨膜电位和pH梯度以及线粒体体积百分比的测定。通过2种不同的方法测量膜电位和pH梯度。[3 H]三苯基甲基磷鎓和[14 C]-硫氰酸根离子的平衡分布在20 - 25 ℃下分别给出计算的表观膜电位为-77.0和-29.6 mV。C;从微电极测量获得-60.8 mV的值。弱酸（[14 C]三甲基乙酸和5，5-二[14 C]甲基-2，4-恶唑烷-二酮）和弱碱（[14 C]二甲胺和[14 C]三甲胺）的平衡分布给出了pH梯度的计算的上限和下限（ΔpH [pH差] = pHe [细胞外pH] - pHi [细胞内pH]）分别为-0.14和-0.21 pH单位。微电极测量结果表明，在7.35-6.85的细胞外pH范围内，细胞内pH值在0.1个pH单位或更小的细胞外pH值内。基于所测量的细胞色素c含量计算的线粒体体积百分比为5.6 ± 0.5。1.2%，并同意5.4 .+-的估计。从25个EM中获得1.1%。细胞能量参数的测量值在其他细胞和灌注器官中发现的范围内。比较微电极和平衡测量的结果，可以估计线粒体膜上的电位和pH梯度（线粒体到细胞质梯度），并表明完整细胞中的跨线粒体膜质子动力不能大于-143 mV。
A method for quantitative evaluation of transmembrane electrical potential and pH gradients across a subcellular compartment in an intact cell is presented. This approach was applied in studies of mouse neuroblastoma C-1300 clone NB41A3, in which the transmembrane electrical potential and pH gradients and the mitochondrial volume percent were determined. Membrane potentials and pH gradients were measured by 2 different methods. Equilibrium distributions of [3H]triphenylmethyl phosphonium and [14C]-thiocyanate ions gave calculated apparent membrane potentials of -77.0 and -29.6 mV, respectively, at 20-25.degree. C; a value of -60.8 mV was obtained from microelectrode measurements. Equilibrium distributions of weak acids ([14C]trimethylacetic acid and 5,5-di[14C]methyl-2,4-oxazolidine-dione) and of weak bases ([14C]dimethylamine and [14C]trimethylamine) gave calculated upper and lower limits of the pH gradient (.DELTA. pH [pH difference] = pHe [extracellular pH] - pHi [intracellular pH]) of -0.14 and -0.21 pH unit, respectively. The microelectrode measurements showed that the intracellular pH is within 0.1 of a pH unit or less of the extracellular pH over the extracellular pH range of 7.35-6.85. The mitochondrial volume percent calculated on the basis of the measured cytochrome c content is 5.6 .+-. 1.2% and agrees with estimates of 5.4 .+-. 1.1% obtained from 25 EM. Measurements of the cellular energetic parameters gave values within the range found in other cells and perfused organs. Comparison of the results of the microelectrode and equilibrium measurements permits estimates of the electrical potential and pH gradients across the mitochondrial membrane (mitochondria-to-cytoplasm gradients) to be made and suggests that the trans-mitochondrial membrane protonmotive force in the intact cell cannot be greater than -143 mV.