MECHANISMS OF NUCLEOSIDE TRANSPORT IN MAMMALIAN CELLS

我们之前的研究已经证明，哺乳动物细胞 有两种不同类型的核苷转运，它们的区别在于 它们对抑制剂的敏感度提高1000倍 并定义了其输运性质。 有一种或另一种(S49小鼠淋巴瘤)的模型细胞系 和Walker 256大鼠癌肉瘤)，或两者(L1210 小鼠白血病)。这一点的延续 该项目将专注于中介蛋白的纯化 NBMPR敏感和耐药的核苷转运，以及 它们的结构和功能特性的比较 蛋白质。获得的单抗将用作 运输蛋白的特定探针，并帮助其 NBMPR与亲和层析的分离纯化 腺苷作为配体也将用于提纯。 学习。这些蛋白质的生物活性将被监测 在脂类转运活性重组的纯化过程中 水泡。重构还将用于比较 纯化蛋白的功能性质。的结构 这些蛋白质将通过肽图谱和测序进行比较 含有底物和NBMPR结合位点的多肽。 从纯化转运研究中获得的信息 蛋白质也将被用来检查结构和取向。 蛋白质以其天然状态存在于 S49和Walker 256细胞的细胞膜。两国之间的关系 对NBMPR敏感和耐药的运输将在 L1210细胞。这些研究将使用遗传和生化 确定这两个运输活动是否 两种分离的不同蛋白质或两种相关蛋白质的性质 相同蛋白质的不同形式。细胞系的转运蛋白(S) 表现出异常的NBMPR结合特性的也将是 审查以更好地了解两国关系 在缓蚀剂结合部位和底物渗透部位之间。它 预计从这些研究中获得的信息将 对理解的长期目标做出重大贡献 核苷转运在哺乳动物中的生理作用 细胞，以及设计使用转运抑制剂的方法来 提高用于癌症的抗代谢药物的选择性 化疗。

Our previous studies have demonstrated that mammalian cells have two distinct types of nucleoside transport that differ by 1000-fold in their sensitivity to the inhibitor nitrobenzylthionosine, and have defined the transport properties of model cell lines having one or the other (S49 mouse lymphoma and Walker 256 rat carcinosarcoma, respectively), or both (L1210 mouse leukemia) of these activities. The continuation of this project will focus on the purification of the proteins mediating NBMPR-sensitive and -resistant nucleoside transport, and comparison of the structural and functional properties of those proteins. Monoclonal antibodies will be obtained to use as specific probes of the transport proteins and to aid in their purification affinity chromatography utilizing NBMPR and adenosine as ligands will also be employed in the purification studies. The biological activity of the proteins will be monitored during purification by reconstitution of transport activity in lipid vesicles. Reconstitution will also be used to compare the functional properties of the purified proteins. The structures of the proteins will be compared by peptide mapping, and sequencing of the peptides bearing the substrate and NBMPR binding sites. The information gained in the study of the purified transport proteins will also be used to examine the structure and orientation of the proteins as they exist in their native state in the membranes of S49 and Walker 256 cells. The relationship between NBMPR-sensitive and - resistant transport will be examined in L1210 cells. These studies will use genetic and biochemical approaches to determine whether the two transport activities are properties of two separate and distinct proteins or two related forms of the same protein. The transport protein(s) of a cell line that exhibits anomolous NBMPR-binding properties will also be examined to gain a better understanding of the relationship between inhibitor binding sites and substrate permeation sites. It is expected that the information gained form these studies will contribute significantly to the long range goals of understanding the physiological roles of nucleoside transport in mammalian cells, and in designing approaches to using transport inhibitors to increase the selectivity of antimetabolites used in cancer chemotherapy.