Lee C W, Goh L B, Tu Y
Department of Physiology, National University of Singapore.
Biochim Biophys Acta. 1995 Aug 31;1268(2):200-8. doi: 10.1016/0167-4889(95)00081-3.
Murine myeloma SP2/0-Ag14 cells possess both nitrobenzylthioinosine (NBMPR)-sensitive and NBMPR-insensitive equilibrative uridine transport systems. No Na(+)-dependent uridine transport system was detected. The NBMPR-insensitive transport system is similarly insensitive to inhibition by dilazep and dipyridamole. Dose-response curve for the inhibition of equilibrative uridine transport by N-ethylmaleimide (NEM), a sulfhydryl reagent, in these cells was biphasic. About 30-40% of the uridine transport was inhibited by NEM at IC50 value of 0.15 mM. The other 60-70% of the transport activity remained insensitive to NEM at concentration as high as 3 mM. The decrease in NBMPR-sensitive uridine transport in the presence of 0.3 mM NEM was due to a 3-fold decrease in transport affinity. Apparent Km values of 500 and 1600 microM and Vmax values of 13 and 12 microM/s were obtained for untreated and NEM-treated cells, respectively. NEM (0.3 mM) has little effect on the Km of NBMPR-insensitive transporter, with apparent Km values of 100 and 110 microM and Vmax values of 3.0 and 2.5 microM/s for untreated and NEM-treated cells, respectively. High sensitivity of NBMPR-sensitive transporter to NEM inhibition was also observed in HL-60 and MCF-7 cells. Decrease in specific 3H-NBMPR equilibrium binding affinity in myeloma cells was observed after treatment with 0.3 mM NEM. Apparent Kd values of 0.32 and 2.3 nM with Bmax values of 48,000 and 44,000 sites/cell were obtained for untreated and NEM-treated cells, respectively. NBMPR, dilazep and dipyridamole at 30 microM, and uridine at 10 mM failed to protect the NBMPR-sensitive transporter against NEM inhibition. It is possible that a critical sulfhydryl residue is closed to substrate binding/transporting site of the NBMPR-sensitive transporter. NEM, a sulfhydryl reagent containing an activated double bond, hinders the affinity of this transporter by forming a stable thiol ether bond with the reactive residue.
小鼠骨髓瘤SP2/0-Ag14细胞同时拥有对硝基苄硫基肌苷（NBMPR）敏感和不敏感的平衡型尿苷转运系统。未检测到依赖Na⁺的尿苷转运系统。对NBMPR不敏感的转运系统对双嘧达莫和地拉卓的抑制同样不敏感。巯基试剂N-乙基马来酰亚胺（NEM）对这些细胞中平衡型尿苷转运的抑制作用的剂量反应曲线呈双相。在IC50值为0.15 mM时，约30%-40% 的尿苷转运被NEM抑制。在高达3 mM的浓度下，另外60%-70% 的转运活性对NEM仍不敏感。在存在0.3 mM NEM的情况下，NBMPR敏感的尿苷转运减少是由于转运亲和力降低了3倍。未处理细胞和经NEM处理的细胞的表观Km值分别为500和1600 μM，Vmax值分别为13和12 μM/s。NEM（0.3 mM）对NBMPR不敏感转运体的Km影响很小，未处理细胞和经NEM处理的细胞的表观Km值分别为100和110 μM，Vmax值分别为3.0和2.5 μM/s。在HL-60和MCF-7细胞中也观察到NBMPR敏感转运体对NEM抑制的高敏感性。在用0.3 mM NEM处理后，观察到骨髓瘤细胞中特异性³H-NBMPR平衡结合亲和力降低。未处理细胞和经NEM处理的细胞的表观Kd值分别为0.32和2.3 nM，Bmax值分别为48,000和44,000位点/细胞。30 μM的NBMPR、双嘧达莫和地拉卓以及10 mM的尿苷均未能保护NBMPR敏感转运体免受NEM抑制。有可能一个关键的巯基残基靠近NBMPR敏感转运体的底物结合/转运位点。NEM是一种含有活化双键的巯基试剂，通过与反应性残基形成稳定硫醚键来阻碍该转运体的亲和力。
