Prabhananda B S, Kombrabail M H
Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai 400 005, India.
Biophys J. 1998 Oct;75(4):1749-58. doi: 10.1016/S0006-3495(98)77616-7.
The decay of the pH difference (DeltapH) across soybean phospholipid vesicular membrane by ionophore A23187 (CAL)-mediated H+/M+ exchange (M+ = Li+, Na+, K+, and Cs+) has been studied in the pH range 6-7.6. The DeltapH in these experiments were created by temperature jump. The observed dependence of DeltapH relaxation rate 1/tau on the concentration of CAL, pH, and the choice of M+ in vesicle solutions lead to the following conclusions. 1) The concentrations of dimers and other oligomers of A23187 in the membrane are small compared to the total concentration of A23187 in the membrane, similar to that in chloroform solutions reported in the literature. 2) In the H+ transport cycle leading to DeltapH decay, the A23187-mediated H+ translocation across the membrane is a fast step, and the rate-limiting step is the A23187-mediated M+ translocation. 3) Even though the monomeric Cal-H is the dominant species translocating H+, Cal-M is not the dominant species translocating M+ (even at concentrations higher than [Cal-H]), presumably because its dissociation rate is much higher than its translocation rate. 4) The pH dependence of 1/tau shows that the dimeric species Cal2LiLi, Cal2NaNa, Cal2KH, and Cal2CsH are the dominant species translocating M+. The rate constant associated with their translocation has been estimated to be approximately 5 x 10(3) s-1. With this magnitude for the rate constants, the dimer dissociation constants of these species in the membrane have been estimated to be approximately 4, 1, 0.05, and 0.04 M, respectively. 5) Contrary to the claims made in the literature, the data obtained in the DeltapH decay studies do not favor the channel mechanism for the ion transport in this system. 6) However, they support the hypothesis that the dissociation of the divalent metal ion-A23187 complex is the rate limiting step of A23187-mediated divalent metal ion transport.
在6 - 7.6的pH范围内，研究了离子载体A23187（CAL）介导的H⁺/M⁺交换（M⁺ = Li⁺、Na⁺、K⁺和Cs⁺）导致大豆磷脂囊泡膜两侧pH差（ΔpH）的衰减情况。这些实验中的ΔpH是通过温度跃升产生的。观察到的ΔpH弛豫速率1/τ对CAL浓度、pH以及囊泡溶液中M⁺选择的依赖性得出了以下结论。1）与膜中A23187的总浓度相比，膜中A23187的二聚体和其他寡聚体的浓度较小，这与文献中报道的氯仿溶液中的情况类似。2）在导致ΔpH衰减的H⁺运输循环中，A23187介导的H⁺跨膜转运是一个快速步骤，限速步骤是A23187介导的M⁺转运。3）尽管单体Cal - H是转运H⁺的主要物种，但Cal - M不是转运M⁺的主要物种（即使在浓度高于[Cal - H]时），推测是因为其解离速率远高于其转运速率。4）1/τ对pH的依赖性表明，二聚体物种Cal₂LiLi、Cal₂NaNa、Cal₂KH和Cal₂CsH是转运M⁺的主要物种。与其转运相关的速率常数估计约为5×10³ s⁻¹。根据这些速率常数的大小，估计这些物种在膜中的二聚体解离常数分别约为4、1、0.05和0.04 M。5）与文献中的说法相反，在ΔpH衰减研究中获得的数据不支持该系统中离子运输的通道机制。6）然而，它们支持这样的假设，即二价金属离子 - A23187复合物的解离是A23187介导的二价金属离子运输的限速步骤。
