13C NMR studies of vitamin C transport and its redox cycling in human erythrocytes
13C NMR studies of vitamin C transport and its redox cycling in human erythrocytes
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DOI：
10.1021/bi970765s
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发表时间：
1998-05-19
期刊：
影响因子：
2.9
通讯作者：
Kuchel, PW
中科院分区：
文献类型：
--
作者：
Himmelreich, U;Drew, KN;Kuchel, PW
发现标记的[1-C-13]-和[2-C-13]-抗坏血酸的C-13 NMR谱包含由人红细胞悬浮液中的细胞内和细胞外群体产生的共振;即，它们显示出“裂峰”现象。这一新的观察结果使得能够随时确定维生素C参与的氧化还原反应的位置，无论是在细胞内部还是外部，并监测化合物进出细胞的运输。因此，抗坏血酸的膜渗透性和脱氢抗坏血酸还原的表观V-max和K-M以非侵入性方式测定。与其他研究不同的是，我们发现了一种不同于葡萄糖转运蛋白的脱氢抗坏血酸转运蛋白，这种转运系统似乎也参与了脱氢抗坏血酸进入细胞的同时还原。第二个还原过程似乎发生在细胞外，通过还原当量通过质膜，发生与铁氰化物的还原。有证据表明，维生素C回收的过程依赖于不同的细胞来源的还原当量。虽然通过膜的运输和还原似乎依赖于糖酵解（NADH），但目前认为，在跨膜电子转移过程中或通过从细胞外运输形成的细胞内脱氢抗坏血酸的还原依赖于NADPH。
C-13 NMR spectra of labeled [1-C-13]- and [2-C-13]ascorbic acid were seen to contain resonances arising from the intra-and extracellular populations in suspensions of human erythrocytes; i.e., they displayed the "split-peak" phenomenon. This new observation enabled the ready determination of the location, whether inside or outside cells, of the redox reactions in which the vitamin C was involved and to monitor the transport of the compounds into and out of the cells. Thus, the membrane permeability of ascorbic acid and the apparent V-max and K-M for the reduction of dehydroascorbic acid were determined in a noninvasive manner. In contrast to other work, evidence was found of a transporter of dehydroascorbic acid which is different from the glucose transporter, This transport system also appeared to be involved in the simultaneous reduction of dehydroascorbic acid on its passage into the cells. A second reduction process appeared to occur extracellularly, by the passage of reducing equivalents through the plasma membrane, as occurs with the reduction of ferricyanide. Evidence is presented that the processes of vitamin C recycling rely on different cellular sources of reducing equivalents. Whereas the transport and reduction via the membrane appeared to be dependent on glycolysis (NADH), the reduction of intracellular dehydroascorbic acid, formed in the process of transmembrane electron transfer or by transport from the outside of the cell, is currently thought to depend on NADPH.