Kolber M A, Haynes D H
Biophys J. 1981 Nov;36(2):369-91. doi: 10.1016/S0006-3495(81)84738-8.
The mechanism for transport of divalent cations across phospholipid bilayers by the ionophore A23187 was investigated. The intrinsic fluorescence of the ionophore was used in equilibrium and rapid-mixing experiments as an indicator of ionophore environment and complexation with divalent cations. The neutral (protonated) form of the ionophore binds strongly to the membrane, with a high quantum yield relative to that in the aqueous phase. The negatively charged form of the ionophore binds somewhat less strongly, with a lower quantum yield, and does not move across the membrane. Complexation of the negatively charged form with divalent cations was measured by the decrease in fluorescence. An apparent rate constant (kapp) for transport of the ionophore across the membrane was determined from the rate of fluorescence changes observed in stopped-flow rapid kinetic experiments. The variation of kapp was studied as a function of pH, temperature, ionophore concentration, membrane lipid composition, and divalent cation concentration and type. Analysis and comparison with equilibrium constants for protonation and complexation show that A23187 and its metal:ionophore complexes bind near the membrane-water interface in the lipid polar-head region. The interfacial reactions occur rapidly, compared with the transmembrane reactions, and are thus in equilibrium during transport. The transport cycle can be described as follows: a 1:1 complex is formed between the membrane bound A23187-(Am-) and the aqueous divalent cation with dissociation constant K1 approximately 4.6 x 10(-4) M. This is in equilibrium with a 1:2 (metal:ionophore) complex (K2 approximately 3.0 x 10(-4) [ionophore/lipid]) that is responsible for transporting the divalent cations across the membrane. The rate constant for translocation of the 1:2 complex is 0.1-0.3 s-1. Dissociation of the complex of the trans side and protonation occur rapidly. The rate constant for translocation of H+ . A23187- is 28 s-1. A theory is presented that is capable of reproducing the kinetic data at any calcium concentration. The cation specificity for ionophore complex transport (kapp), determined at low ionophore concentration for a series of divalent cations, was found to be proportional to the equilibrium constant for 1:1 complexation. The order of ion specificity for these processes was found to be Ca2+ greater than Mg2+ greater Sr2+ greater than Ba2+. Interactions with Na+ were not observed. Maximal values of kapp were observed for vesicles prepared from pure dimyristoyl phosphatidylcholine. Inclusion of phosphatidyl ethanolamine, phosphatidic acid, or dipalmatoyl phosphatidylcholine resulted in lower values of kapp. Calcium transport by A23187 is compared with that of X537A, and it is shown that the former is 67-fold faster. The difference in rates is due to differences in the ability of each ionophore to form a 1:2 complex from a 1:1 complex.
研究了离子载体A23187介导二价阳离子跨磷脂双分子层转运的机制。在平衡和快速混合实验中，利用离子载体的固有荧光作为离子载体环境以及与二价阳离子络合的指标。离子载体的中性（质子化）形式与膜紧密结合，相对于水相具有较高的量子产率。离子载体的带负电荷形式结合力稍弱，量子产率较低，且不穿过膜。通过荧光降低来测量带负电荷形式与二价阳离子的络合。根据停流快速动力学实验中观察到的荧光变化速率，确定离子载体跨膜转运的表观速率常数（kapp）。研究了kapp随pH、温度、离子载体浓度、膜脂质组成以及二价阳离子浓度和类型的变化。与质子化和络合平衡常数的分析和比较表明，A23187及其金属：离子载体络合物在脂质极性头部区域的膜 - 水界面附近结合。与跨膜反应相比，界面反应发生迅速，因此在转运过程中处于平衡状态。转运循环可描述如下：膜结合的A23187 - （Am - ）与水相二价阳离子形成1:1络合物，解离常数K1约为4.6×10（ - 4）M。这与负责将二价阳离子跨膜转运的1:2（金属：离子载体）络合物（K2约为3.0×10（ - 4）[离子载体/脂质]）处于平衡状态。1:2络合物转运的速率常数为0.1 - 0.3 s - 1。跨侧络合物的解离和质子化迅速发生。H +.A23187 - 的转位速率常数为28 s - 1。提出了一种能够在任何钙浓度下重现动力学数据的理论。在低离子载体浓度下，针对一系列二价阳离子测定的离子载体络合物转运的阳离子特异性（kapp）与1:1络合的平衡常数成正比。发现这些过程的离子特异性顺序为Ca2 + ＞Mg2 + ＞Sr2 + ＞Ba2 + 。未观察到与Na + 的相互作用。由纯二肉豆蔻酰磷脂酰胆碱制备的囊泡观察到kapp的最大值。加入磷脂酰乙醇胺、磷脂酸或二棕榈酰磷脂酰胆碱会导致kapp值降低。将A23187介导的钙转运与X537A的进行比较，结果表明前者快67倍。速率差异是由于每种离子载体从1:1络合物形成1:2络合物的能力不同。
