Molecular aspect of ascorbate transport system and its application for evaluation of toxicity of envioronmental pollutants

(1)为了阐明抗坏血酸转运系统的分子机制，采用chaps溶解法制备大鼠肾膜，研究了抗坏血酸的膜结合活性。添加抗坏血酸和高剂量脱氢抗坏血酸可以有效抑制这种结合活性，而葡萄糖和葡萄糖转运抑制剂则没有抑制作用。此外，该制剂被发现结合抗坏血酸的还原形式，强烈支持抗坏血酸特异性结合蛋白的存在，该蛋白被认为是其在质膜上运输系统的成员。(2)该结合蛋白经凝胶过滤分离，活性保持良好，分子量估计在100kDa左右。然而，到目前为止，还没有任何色谱法进行进一步的纯化，导致活性显著降低。对于其纯化，需要建立条件来重建结合络合物。(3)本项目首次发现胃中抗坏血酸的分泌是通过胆碱能受体介导的，这一机制是胃中抗坏血酸具有重要生理意义的机制。此外，抗坏血酸在小肠中的吸收效率也受到尼古丁的影响。这些结果表明，环境污染物动态影响抗坏血酸在肠道中的运输系统。为了评价抗坏血酸对污染物的毒性，需要进一步研究建立抗坏血酸细胞特异性摄取系统。

(1) In order to clarify the molecular aspect of ascorbate transport system, membraneous ascorbatebinding activity was studied by using CHAPS-solubilized preparation of rat kidney membranes. This binding activity was effectively inhibited by the addition of ascorbate and a high dose of dehydroascorbate, whereas glucose and glucose-transport inhibitors showed no inhibition.Moreover, this preparation was found to bind the reduced form of ascorbate, strongly supporting the presence of an ascorbate-specific binding protein which is considered to be a member of its transport system on the plasma membranes. (2) This binding protein was separated by gel filtration with a good retention of activity and its molecular weight was estimated to be about 100kDa. However, further purification was not achieved by any chromatography until now, resulting in remarkable decrease of activity. For its purification, it is necessary to establish the conditions to reconstitute the binding complex. (3) In this project we have found for the first time that gastric ascorbate secretion is mediated via cholinergic receptor and this mechanism is responsible for physiological importance of ascorbate in the stomach. In addition, the absorption efficiency of ascorbate in the small intestine was found to be influenced by nicotine. These results suggest that environmental pollutants dynamically affect the transport system of ascorbate in the gut. We need further investigation to establish ascorbate-specific uptake system of cell line for evaluation of toxicity by pollutants.