Spiess I, Wang J, Benz R, Zimmermann U
Lehrstuhl für Biotechnologie, Universität Würzburg, Germany.
Biochim Biophys Acta. 1993 Jun 18;1149(1):93-101. doi: 10.1016/0005-2736(93)90029-y.
The effect of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) on the Cl(-)-transport system located in the plasmalemma of cells of the giant marine alga Valonia utricularis was studied by using the charge pulse relaxation technique. Analysis of the biphasic relaxation patterns in terms of the kinetic model published previously (Wang, J., Wehner, G., Benz, R. and Zimmermann, U. (1991) Biophys. J. 59, 235-248) demonstrated that extracellular DIDS dramatically reduced the translocation rate, KAS, of the Cl(-)-carrier complex (maximal inhibition 79%). The translocation rate of the free carrier molecules, KS, as well as the total surface concentration of the carrier, No, were not affected. A Hill-plot of DIDS inhibition on KAS yielded an half-maximal inhibition concentration (IC50) of 3.9 x 10(-5) M and Hill-coefficient of 1.61, suggesting a co-operative binding of the inhibitors to the Cl(-)-carrier. The maximal inhibition of DIDS was dependent on the extracellular Cl(-)-concentration. This inhibition was not competitive to chloride, since it increased and did not decrease with increasing chloride concentration. The DIDS effect decreased with increasing pH-value (investigated pH range between 6.5 and 10). Intravascular DIDS or SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid) had no effect on the biphasic voltage relaxation pattern. These results showed that the binding sites of DIDS must be located on the outer surface of the plasmalemma of V. utricularis and, in turn, supported previous conclusions that the Cl(-)-carrier (which is assumed to be part of the turgor-pressure-sensing mechanism) is only located in the outer membrane.
利用电荷脉冲弛豫技术研究了阴离子转运抑制剂4,4'-二异硫氰基芪-2,2'-二磺酸（DIDS）对大型海藻瓦氏马尾藻细胞质膜中Cl⁻转运系统的影响。根据先前发表的动力学模型（Wang, J., Wehner, G., Benz, R.和Zimmermann, U. (1991) Biophys. J. 59, 235 - 248）对双相弛豫模式进行分析表明，细胞外DIDS显著降低了Cl⁻载体复合物的转运速率KAS（最大抑制率79%）。游离载体分子的转运速率KS以及载体的总表面浓度No不受影响。DIDS对KAS抑制作用的希尔图得出半最大抑制浓度（IC50）为3.9×10⁻⁵ M，希尔系数为1.61，表明抑制剂与Cl⁻载体协同结合。DIDS的最大抑制作用取决于细胞外Cl⁻浓度。这种抑制作用对氯离子不具有竞争性，因为它随着氯离子浓度的增加而增加而非降低。DIDS的作用随着pH值的升高而降低（研究的pH范围在6.5至10之间）。血管内注射DIDS或SITS（4-乙酰氨基-4'-异硫氰基芪-2,2'-二磺酸）对双相电压弛豫模式没有影响。这些结果表明，DIDS的结合位点一定位于瓦氏马尾藻质膜的外表面，进而支持了先前的结论，即Cl⁻载体（假定为膨压传感机制的一部分）仅位于外膜中。
