Crawford C R, Ng C Y, Ullman B, Belt J A
Department of Biochemical and Clinical Pharmacology, St. Jude Children's Research Hospital, Memphis, TN 38101.
Biochim Biophys Acta. 1990 May 24;1024(2):289-97. doi: 10.1016/0005-2736(90)90357-t.
The major nucleoside transporter of the human T leukemia cell line CEM has been identified by photoaffinity labeling with the transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR). The photolabeled protein migrates on SDS-PAGE gels as a broad band with a mean apparent molecular weight (75,000 +/- 3000) significantly higher than that reported for the nucleoside transporter in human erythrocytes (55,000) (Young et al. (1983) J. Biol. Chem. 258, 2202-2208). However, after treatment with endoglycosidase F to remove carbohydrate, the NBMPR-binding protein in CEM cells migrates as a sharp peak with an apparent molecular weight (47,000 +/- 3000) identical to that reported for the deglycosylated protein in human erythrocytes (Kwong et al. (1986) Biochem. J. 240, 349-356). It therefore appears that the difference in the apparent molecular weight of the NBMPR-sensitive nucleoside transporter between the CEM cell line and human erythrocytes is a result of differences in glycosylation. The NBMPR-binding protein from CEM cells has been solubilized with 1% octyl glucoside and reconstituted into phospholipid vesicles by a freeze-thaw sonication technique. Optimal reconstitution of uridine transport activity was achieved using a sonication interval of 5 to 10 s and lipid to protein ratios of 60:1 or greater. Under these conditions transport activity in the reconstituted vesicles was proportional to the protein concentration and was inhibited by NBMPR. Omission of lipid or protein, or substitution of a protein extract prepared from a nucleoside transport deficient mutant of the CEM cell line resulted in vesicles with no uridine transport activity. The initial rate of uridine transport, in the vesicles prepared with CEM protein, was saturable with a Km of 103 +/- 11 microM and was inhibited by adenosine, thymidine and cytidine. The Km for uridine and the potency of the other nucleosides as inhibitors of uridine transport (adenosine greater than thymidine greater than cytidine) were similar to intact cells. Thus, although the nucleoside transporter of CEM cells has a higher molecular weight than the human erythrocyte transporter, it exhibits typical NBMPR-sensitive nucleoside transport activity both in the intact cell and when reconstituted into phospholipid vesicles.
通过用转运抑制剂硝基苄基巯基嘌呤核糖苷（NBMPR）进行光亲和标记，已鉴定出人类T白血病细胞系CEM的主要核苷转运体。光标记的蛋白在SDS-PAGE凝胶上迁移为一条宽带，平均表观分子量为（75,000±3000），显著高于人类红细胞中核苷转运体的报道值（55,000）（Young等人，（1983年）《生物化学杂志》258, 2202 - 2208）。然而，用内切糖苷酶F处理以去除碳水化合物后，CEM细胞中的NBMPR结合蛋白迁移为一个尖锐峰，表观分子量为（47,000±3000），与人类红细胞中去糖基化蛋白的报道值相同（Kwong等人，（1986年）《生物化学杂志》240, 349 - 356）。因此，CEM细胞系和人类红细胞之间NBMPR敏感核苷转运体表观分子量的差异似乎是糖基化差异的结果。来自CEM细胞的NBMPR结合蛋白已用1%辛基葡糖苷溶解，并通过冻融超声技术重构到磷脂囊泡中。使用5至10秒的超声处理间隔和60:1或更高的脂质与蛋白比例，可实现尿苷转运活性的最佳重构。在这些条件下，重构囊泡中的转运活性与蛋白浓度成正比，并被NBMPR抑制。省略脂质或蛋白，或用从CEM细胞系的核苷转运缺陷突变体制备的蛋白提取物替代，会导致囊泡没有尿苷转运活性。用CEM蛋白制备的囊泡中尿苷转运的初始速率是可饱和的，Km为103±11微摩尔，并且被腺苷、胸苷和胞苷抑制。尿苷的Km以及其他核苷作为尿苷转运抑制剂的效力（腺苷＞胸苷＞胞苷）与完整细胞相似。因此，尽管CEM细胞的核苷转运体分子量高于人类红细胞转运体，但它在完整细胞中以及重构到磷脂囊泡中时都表现出典型的NBMPR敏感核苷转运活性。
