MECHANISMS OF NUCLEOSIDE TRANSPORT IN MAMMALIAN CELLS

我们之前的研究表明哺乳动物细胞 有两种不同类型的核苷转运， 1000-对抑制剂的敏感性增加一倍 nitrobenzylthionosine，并已确定的运输性能 具有一种或另一种的模型细胞系（S49小鼠淋巴瘤 和步行者256大鼠癌肉瘤），或两者（L1210 小鼠白血病）的这些活性。 继续开展这一 该项目将集中在蛋白质的纯化介导 NBMPR敏感和耐药核苷转运，和 比较了它们的结构和功能特性， proteins. 将获得单克隆抗体，用作 转运蛋白的特异性探针， 利用NBMPR的纯化亲和色谱法和 腺苷作为配体也将用于纯化 问题研究 将监测蛋白质的生物活性 在通过重建脂质中的转运活性的纯化过程中 囊泡 复溶也将用于比较 纯化蛋白质的功能特性。 的结构 蛋白质将通过肽图谱和测序进行比较 具有底物和NBMPR结合位点的肽。 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方向 蛋白质的自然状态 S49和步行者256细胞膜。 的关系 NBMPR敏感和耐药转运将在 L1210细胞。 这些研究将使用遗传和生化技术 确定这两项运输活动是否 两种不同的蛋白质或两种相关的蛋白质 同一种蛋白质。 细胞系的转运蛋白 表现出异常NBMPR结合特性的药物也将被 为了更好地理解 在抑制剂结合位点和底物渗透位点之间。 它 预计从这些研究中获得的信息将 对长期目标的理解做出了重大贡献 哺乳动物核苷转运生理作用 细胞，并在设计使用转运抑制剂的方法， 增加用于癌症的抗代谢物的选择性 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.