VITAMINS B2 AND B6 TRANSPORT BY RENAL AND HEPATIC CELLS

虽然有广泛的信息的要求，新陈代谢， 辅酶的功能的B-复合维生素，有一个知识匮乏 关于这些必需营养素在体内运输的方式， 哺乳动物细胞大多数早期的研究都集中在胃肠道 摄取、循环转运和最终在靶器官中的总体分布 细胞很少注意到所涉及的具体机制 从不同哺乳动物中摄取（和排出）几种B族维生素 细胞类型。我们的实验室已经获得了 分离的肝细胞摄取某些B族维生素的不同方法 来自大鼠肝脏和来自大鼠肾脏的肾近端小管细胞。这些 研究表明，核黄素和维生素B6进入肝脏， 并且肾细胞被促进并表现出特异性;因此， 指出了血浆-膜载体系统。其他人最近的工作， 荧光技术已经鉴定出了 肝细胞质膜我们实验室的初步工作， 亲和层析分离出B6结合蛋白 来自肾细胞的管腔质膜。 本提案旨在阐明具体机制， 核黄素和维生素B进入和离开肝和肾细胞。的 负责促进血浆转运的蛋白质 这些细胞的膜将通过破裂活细胞而分离， 密度梯度离心以差异选择质膜， 蛋白质组分的增溶和色谱选择 那些对维生素有最大亲和力的。的结合蛋白 将表征分子性质（M.W.，K（a）代表维生素， 特异性等）重组成囊泡， 运输得到加强。抗体会被制造出来，在某些情况下， 荧光标记，以验证功能和定位的 质膜、囊泡和细胞内的维生素结合蛋白。 拟议的研究将大大扩展对如何 核黄素和维生素B6进入和离开肝脏和肾脏细胞 差异参与代谢转化和排泄。此外，委员会认为， 正常和异常细胞之间维生素转运的潜在差异， 无论是缺乏维生素还是肿瘤， 关于这些病理状态下的营养利用。

Though there is extensive information on the requirements, metabolism, and coenzymic functions of B-complex vitamins, there is a paucity of knowledge concerning the means by which such essential nutrients are transported in mammalian cells. Most earlier studies have focused on gastrointestinal uptake, circulatory transport, and the ultimate gross disposition in target cells. Little attention has been given to the specific mechanisms involved in uptake (and efflux) of several B vitamins from the diverse mammalian cell types. Our laboratory has acquired general information on the different means for uptake of certain B vitamins by hepatocytes isolated from rat liver and by renal proximal tubular cells from rat kidney. These studies reveal that the entry of riboflavin and vitamin B6 in both liver and kidney cells is facilitated and exhibits specificity; hence, plasma-membrane carrier systems were indicated. Recent work by others using fluorescence technique has identified flavin-binding protein(s) in the hepatocyte plasma membrane. Preliminary work in our laboratory using affinity chromatography has led to the isolation of B6-binding proteins from the luminal plasma membranes of renal cells. The present proposal is to elucidate the specific mechanisms by which riboflavin and vitamin B, enter and exit both liver and kidney cells. The proteins responsible for facilitating transport through the plasma membranes of such cells will be isolated by rupture of viable cells, density-gradient centrifugation to differentially select plasma membranes, solubilization of protein components, and chromatographic selection of those that have greatest affinity for the vitamin. The binding proteins will be characterized for molecular properties (M.W., K(a) for vitamin, specificity, etc.) and reconstituted into vesicles to see to what extent transport is enhanced. Antibodies will be made and in some cases fluorescence-labeled to verify function and localization of the vitamin-binding proteins within the plasma-membranes, vesicles, and cells. The proposed research will significantly expand understanding of how riboflavin and vitamin B6 enter and exit liver and kidney cells differentially involved in metabolic conversion and excretion. Moreover, potential differences in vitamin transport among normal and abnormal cells, either vitamin-deficient or tumor, will provide critical knowledge concerning nutrient utilization in these pathological states.