VITAMINS B2 AND B6 TRANSPORT BY RENAL AND HEPATIC CELLS

尽管有大量关于需求、新陈代谢和 B-复合维生素的辅酶功能，目前知之甚少 关于这些必需的营养物质在 哺乳动物细胞。大多数早期的研究都集中在胃肠道。 靶向摄取、循环转运和最终总处置 细胞。很少有人注意到涉及的具体机制。 从不同的哺乳动物摄取(和排出)几种B族维生素 单元类型。我们的实验室已经获得了有关该病毒的一般信息。 分离肝细胞摄取某些B族维生素的不同途径 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进入两个肝脏 肾脏细胞被促进并表现出特异性；因此， 指出了质膜载体系统。其他人最近使用的工作 荧光技术鉴定黄素结合蛋白(S) 肝细胞质膜。我们实验室的初步工作是使用 亲和层析使B6结合蛋白得以分离 来自肾细胞的管腔质膜。 目前的建议是阐明通过哪些具体机制 核黄素和维生素B进入和离开肝脏和肾脏细胞。这个 负责促进通过血浆转运的蛋白质 这样的细胞膜将通过活细胞的破裂而分离， 密度梯度离心法以区别选择质膜， 蛋白质组分的增溶和层析筛选 那些对维生素有最大亲和力的人。结合蛋白 将表征维生素的分子性质(M.W.，K(A)， 专属性等。)并重组成囊泡，看看能达到什么程度 交通得到了加强。会产生抗体，在某些情况下 荧光标记以验证细胞的功能和定位 质膜、囊泡和细胞中的维生素结合蛋白。 这项拟议的研究将极大地扩大对 核黄素和维生素B6进出肝和肾细胞 不同地参与代谢转换和排泄。此外， 正常和异常细胞之间维生素转运的潜在差异， 无论是维生素缺乏还是肿瘤，都将提供关键的知识 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.