Yamato I, Anraku Y
Department of Biology, Faculty of Science, University of Tokyo, Japan.
J Membr Biol. 1990 Mar;114(2):143-51. doi: 10.1007/BF01869095.
The proton and sodium ion dependences of the proline binding and transport activities of the proline carrier in Escherichia coli were investigated in detail. The binding activity in cytoplasmic membrane vesicles from a carrier over-producing strain (PT21/pTMP5) was absolutely dependent on the presence of Na+, but did not necessarily require protonation of the carrier, in contrast to the model previously reported (Mogi, T., Anraku, Y. 1984. J. Biol. Chem. 259:7797-7801). Based on this and previous observations, we propose a modified model of the proline binding reaction of the proline carrier, in which a proton is supposed to be a regulatory factor for the binding activity. The apparent Michaelis constant of proline (Kt) of the transport activity of cytoplasmic membrane vesicles from the wild type E. coli strain driven by a respiratory substrate, ascorbate, showed dependence on a low concentration of sodium ion. The Michaelis constant of sodium ion for transport (KtNa) was estimated to be 25 microM. The proline transport activities in membrane vesicles and intact cells were modulated by H+ concentration, the inhibitory effect of protons (pKa approximately equal to 6) being similar to that observed previously (Mogi, T., Anraku, Y. 1984. J. Biol. Chem. 259:7802-7806). Based on these observations and the modified model of substrate binding to the proline carrier, a model of the proline/Na+ symport mechanism is proposed, in which a proton is postulated to be a regulatory factor of the transport activity.
我们详细研究了大肠杆菌中脯氨酸载体的脯氨酸结合及转运活性对质子和钠离子的依赖性。与之前报道的模型（Mogi, T., Anraku, Y. 1984. J. Biol. Chem. 259:7797 - 7801）相反，来自载体过量表达菌株（PT21/pTMP5）的细胞质膜囊泡中的结合活性绝对依赖于钠离子的存在，但不一定需要载体的质子化。基于此及之前的观察结果，我们提出了脯氨酸载体脯氨酸结合反应的修正模型，其中质子被认为是结合活性的调节因子。由呼吸底物抗坏血酸驱动的野生型大肠杆菌菌株的细胞质膜囊泡转运活性的脯氨酸表观米氏常数（Kt）显示出对低浓度钠离子的依赖性。钠离子转运的米氏常数（KtNa）估计为25微摩尔。膜囊泡和完整细胞中的脯氨酸转运活性受H⁺浓度调节，质子的抑制作用（pKa约等于6）与之前观察到的相似（Mogi, T., Anraku, Y. 1984. J. Biol. Chem. 259:7802 - 7806）。基于这些观察结果以及底物与脯氨酸载体结合的修正模型，我们提出了脯氨酸/钠离子同向转运机制的模型，其中质子被假定为转运活性的调节因子。
