Bogucka K, Wroniszewska A, Bednarek M, Duszyński J, Wojtczak L
Department of Cellular Biochemistry, Nencki Institute of Experimental Biology, Warsaw, Poland.
Biochim Biophys Acta. 1990 Feb 22;1015(3):503-9. doi: 10.1016/0005-2728(90)90084-h.
Ehrlich ascites tumour cells were treated with digitonin so that they became permeable for low-molecular-weight compounds but, at certain concentrations of digitonin, retained most of their cytoplasmic proteins. Respiration of mitochondria with exogenous substrates and their membrane potential could thus be measured in situ by means of oxygen electrode and tetraphenylphosphonium-sensitive electrode, respectively. The results were compared with data from similar measurements on mitochondria isolated from such digitonin-permeabilized cells. Isolated mitochondria and mitochondria in situ oxidized succinate at similar rates and developed membrane potential of comparable magnitude. Both preparations also exhibited an identical nonlinear relationship between resting state respiration (titrated with a respiratory inhibitor) and the membrane potential. In the cells permeabilized with low concentrations of digitonin (i.e., retaining most of cytoplasmic proteins) and suspended in medium containing NaCl and other major anions and cations at concentrations close to those in mammalian plasma, anaerobiosis did not produce a decrease in the mitochondrial membrane potential, which was collapsed only after a subsequent addition of oligomycin. In this medium, glucose had little effect on either respiration or the membrane potential.
用洋地黄皂苷处理艾氏腹水瘤细胞，使其对低分子量化合物具有通透性，但在一定浓度的洋地黄皂苷作用下，细胞仍保留大部分细胞质蛋白。因此，可分别通过氧电极和四苯基鏻敏感电极原位测量线粒体利用外源底物的呼吸作用及其膜电位。将这些结果与从经洋地黄皂苷通透处理的细胞中分离得到的线粒体进行类似测量的数据进行比较。分离得到的线粒体和原位线粒体以相似的速率氧化琥珀酸，并产生相当大小的膜电位。两种制剂在静息状态呼吸（用呼吸抑制剂滴定）和膜电位之间也呈现相同的非线性关系。在用低浓度洋地黄皂苷通透处理的细胞（即保留大部分细胞质蛋白）中，将细胞悬浮于含有氯化钠及其他主要阴离子和阳离子、浓度接近哺乳动物血浆的培养基中，无氧条件下线粒体膜电位并未降低，仅在随后添加寡霉素后才崩溃。在这种培养基中，葡萄糖对呼吸作用或膜电位几乎没有影响。
