Vidaver G A, Shepherd S L, Lagow J B, Wiechelman K J
Biochim Biophys Acta. 1976 Sep 7;443(3):494-514. doi: 10.1016/0005-2736(76)90468-5.
The influence of a Donnan effect on the transport of glycine by hemolysed and restored pigeon red cells was examined. The Donnan effect was produced by replacing Cl- with 2,4-toluenedisulfonate or glutamate. The effects of the associated membrane potential and inside-outside pH difference on glycine entry and exit rates were examined. The effects of pH on entry and exit rates in the absence of a Donnan effect were also examined. In the absence of a Donnan effect, Na+-dependent glycine entry requires the protonated form of a group with a pKapp of 7.9 and the deprotonated form of another group with a pKapp of 6.8. Neither of these are required for exit but the deprotonated form of a group(s) with a pKapp of 6.2 is required. The pK 7.9 group and pK 6.2 group probably react with H+ at the inner face of the membrane and the pK 6.8 group probably reacts at the outer face. The V for glycine entry was determined for cells with their Cl- largely replaced by toluenedisulfonate and without such replacement. Between pH 6.1 and 7, the ratio of the respective V values, VT/VC1, was 1.5-1.7. VT/VC1 rose above pH 7 to near 4 at pH 8.3. At pH 6.9, with glutamate replacing cell Cl-, the analogous ratio (VGlu/VC1) was 1.7. The increase of VT/VC1 above pH 7 could be quantitatively accounted for by the increase in cell [H+]/medium [H+] caused by the Donnan effect together with the assumption that the pK 7.9 group reacts with H+ at the inner face of the membrane. When cell Cl- was replaced by toluenedisulfonate or glutamate there was a drop in the term in the glycine Km describing Na+ dependence of glycine entry. When cell Cl- was replaced by toluenedisulfonate therewas a rise in the Na+-independent term in the glycine entry Km. By replacing varying amounts of cell Cl- with either toluenedisulfonate or glutamate, plots were obtained of entry rates vs. the cell [Cl-]/ medium [Cl-] ratio consistent with the assumption that the Donnan-induced membrane potential acts on a "moving" charge. Glycine exit was only slightly accelerated by trans-toluenedisulfonate. The ratio, exit rate into toluenedisulfonate medium/exit rate into Cl- medium rose with decreasing pH. This rise could be accounted for by a Donnan-induced inside-outside pH difference which affects a pKapp 6.2 group reacting with internal H+. The observed influences of the Donnan effect on V (glycine entry), on both components of Km (glycine entry), on the shape of the plot of glycine entry rate vs. the cell [Cl-]/medium [Cl-] ratio and on glycine exit all fit the assumptions that when the empty porter reorients, one unit of negative charge accompanies it "across" the membrane and that no other steps involve charge movement. The properties of the system seem inconsistent with a translational ("ferry boat") mobile carrier.
研究了唐南效应（Donnan effect）对溶血及恢复后的鸽红细胞转运甘氨酸的影响。通过用2,4 - 甲苯二磺酸盐或谷氨酸替代Cl⁻来产生唐南效应。研究了相关膜电位和内外pH差值对甘氨酸进出速率的影响。还研究了在不存在唐南效应时pH对进出速率的影响。在不存在唐南效应时，Na⁺依赖的甘氨酸进入需要一个表观解离常数（pKapp）为7.9的基团的质子化形式和另一个pKapp为6.8的基团的去质子化形式。这些对于甘氨酸的流出都不是必需的，但需要一个pKapp为6.2的基团（或多个基团）的去质子化形式。pK 7.9的基团和pK 6.2的基团可能在膜的内表面与H⁺反应，而pK 6.8的基团可能在膜的外表面反应。测定了Cl⁻大部分被甲苯二磺酸盐替代的细胞和未被替代的细胞的甘氨酸进入速率（V）。在pH 6.1至7之间，各自的V值之比VT/VC1为1.5 - 1.7。VT/VC1在pH 7以上升高，在pH 8.3时接近4。在pH 6.9时，用谷氨酸替代细胞内的Cl⁻，类似的比值（VGlu/VC1）为1.7。VT/VC1在pH 7以上的增加可以通过唐南效应引起的细胞内[H⁺]/培养基[H⁺]的增加以及假设pK 7.9的基团在膜的内表面与H⁺反应来定量解释。当细胞内的Cl⁻被甲苯二磺酸盐或谷氨酸替代时，描述甘氨酸进入对Na⁺依赖性的甘氨酸米氏常数（Km）项下降。当细胞内的Cl⁻被甲苯二磺酸盐替代时，甘氨酸进入Km中的非Na⁺依赖性项升高。通过用甲苯二磺酸盐或谷氨酸替代不同量的细胞内Cl⁻，得到了进入速率与细胞[Cl⁻]/培养基[Cl⁻]比值的关系图，这与唐南诱导的膜电位作用于“移动”电荷的假设一致。反式甲苯二磺酸盐仅略微加速了甘氨酸的流出。流出到甲苯二磺酸盐培养基中的速率/流出到Cl⁻培养基中的速率之比随着pH降低而升高。这种升高可以通过唐南诱导的内外pH差值来解释，该差值影响与内部H⁺反应的pKapp 6.2的基团。观察到的唐南效应对V（甘氨酸进入）、对Km（甘氨酸进入）的两个组分、对甘氨酸进入速率与细胞[Cl⁻]/培养基[Cl⁻]比值的关系图的形状以及对甘氨酸流出的影响都符合以下假设：当空载体重新定向时，一个单位的负电荷伴随其“穿过”膜，并且没有其他步骤涉及电荷移动。该系统的特性似乎与平移式（“渡船”）移动载体不一致。
