Vincenzini M T, Iantomasi T, Favilli F
Department of Biochemical Sciences, University of Florence, Italy.
Biochim Biophys Acta. 1989 Dec 11;987(1):29-37. doi: 10.1016/0005-2736(89)90451-3.
We characterized glutathione transport in brush-border membrane vesicles (BBMV) that were prepared from rabbit small intestine in which gamma-glutamyl transpeptidases (gamma-glutamyltransferases, EC 2.3.2.2) had been inactivated by a specific affinity-labeling reagent (AT125). Intact GSH transport was strongly increased by the presence of Na+, K+, LI+, Ca2+ and Mn2+ and, of all these, the Ca2+ activation effect was prevalent. This cation effect was selective and catalytic but not energetic; Vmax obtained in the presence of both Na+ and Ca2+ was about 6-times higher than it was in their absence, while Km did not change. Moreover, these cations almost completely eliminated GSH binding on the membrane surface. Na+ activation cannot be explained as a stimulation effect on the Na+-H+ antiport system, since a GSH proton-driven transport was excluded. We determined a pH optimum (7.5), while low or high extravesicular pH values diminished the GSH uptake rate. The Ca2+ effect on GSH transport, when an electrical potential difference was imposed across BBMV, was different from that of monovalent cations. Indeed, experiments performed by valinomycin-induced K+ diffusion potential or by anion substitution showed that the GSH transport system was an electroneutral process in the presence of Na+ or K+, but that it was electrogenic in the presence of Ca2+ or in the absence of extravesicular cations. These results suggest that GSH is also cotransported with these cations, without its accumulation inside vesicles. Moreover, since GSH is negatively charged, the effect of pH changes and of cation activation on GSH transport is arguably mediated by changes in the ionization state of certain groups as the carrier site and of GSH itself, indicating the electrostatic nature of GSH binding sites on the transporter. The high Ca2+ activation effect is perhaps also partly due to fluidity changes in the lipoproteic microenvironment of the GSH transporter. Moreover, this transport system has high affinity with GSH, given the low Km value (17 microM) and the fact that it was only inhibited by GSH S-derivatives and by GSH monoethyl ester, which probably share the same transport system.
我们对从兔小肠制备的刷状缘膜囊泡（BBMV）中的谷胱甘肽转运进行了表征，其中γ-谷氨酰转肽酶（γ-谷氨酰转移酶，EC 2.3.2.2）已被一种特异性亲和标记试剂（AT125）灭活。Na⁺、K⁺、Li⁺、Ca²⁺和Mn²⁺的存在会强烈增加完整谷胱甘肽的转运，在所有这些离子中，Ca²⁺的激活作用最为显著。这种阳离子效应具有选择性和催化性，但不涉及能量；在同时存在Na⁺和Ca²⁺的情况下获得的Vmax比不存在它们时高约6倍，而Km不变。此外，这些阳离子几乎完全消除了谷胱甘肽在膜表面的结合。Na⁺激活不能解释为对Na⁺-H⁺反向转运系统的刺激作用，因为谷胱甘肽的质子驱动转运被排除在外。我们确定了最适pH值（7.5），而囊泡外低或高的pH值会降低谷胱甘肽的摄取速率。当在BBMV上施加电位差时，Ca²⁺对谷胱甘肽转运的影响与单价阳离子不同。实际上，通过缬氨霉素诱导的K⁺扩散电位或阴离子替代进行的实验表明，在存在Na⁺或K⁺的情况下，谷胱甘肽转运系统是一个电中性过程，但在存在Ca²⁺或不存在囊泡外阳离子的情况下是生电的。这些结果表明，谷胱甘肽也与这些阳离子协同转运，而不会在囊泡内积累。此外，由于谷胱甘肽带负电荷，pH变化和阳离子激活对谷胱甘肽转运的影响可以说是由载体位点和谷胱甘肽本身某些基团的电离状态变化介导的，这表明转运体上谷胱甘肽结合位点的静电性质。高Ca²⁺激活效应可能也部分归因于谷胱甘肽转运体脂蛋白微环境中的流动性变化。此外，鉴于低Km值（17μM）以及它仅被谷胱甘肽S-衍生物和谷胱甘肽单乙酯抑制这一事实，该转运系统对谷胱甘肽具有高亲和力，这两者可能共享相同的转运系统。
