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方面，用CHAPS增溶的大鼠肾膜制备物研究了膜抗坏血酸结合活性。这种结合活性被有效地抑制了添加抗坏血酸和高剂量的脱氢抗坏血酸，而葡萄糖和葡萄糖转运抑制剂显示没有inhibit.Further，这种制剂被发现绑定的抗坏血酸的还原形式，强烈支持存在的抗坏血酸特异性结合蛋白，这被认为是其运输系统的质膜上的一个成员。(2)该结合蛋白经凝胶过滤分离后，其分子量约为100kDa，活性保留良好。然而，迄今为止没有通过任何层析实现进一步纯化，导致活性显著降低。对于其纯化，有必要建立重组结合复合物的条件。(3)在本项目中，我们首次发现胃抗坏血酸分泌是通过胆碱能受体介导的，这种机制是抗坏血酸在胃中的生理重要性的原因。此外，抗坏血酸在小肠中的吸收效率被发现受到尼古丁的影响。这些结果表明，环境污染物动态影响肠道中抗坏血酸的运输系统。因此，建立抗坏血酸特异性细胞摄取系统用于污染物毒性评价尚需进一步研究。

(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.