Dahlig-Harley E, Eilam Y, Paterson A R, Cass C E
Biochem J. 1981 Nov 15;200(2):295-305. doi: 10.1042/bj2000295.
Nitrobenzylthioinosine (NBMPR) binds reversibly, but with high affinity (Kd 0.1--1.2 nM), to inhibitory sites on nucleoside-transport elements of the plasma membrane in a variety of animal cells. The present study explored relationships in HeLa cells between NBMPR binding and inhibition of uridine transport. The Km value for inward transport of uridine by HeLa cells in both suspension and monolayer culture was about 0.1 mM. The affinity of the transport-inhibitory sites for uridine (Kd 1.7 mM), inosine (Kd 0.4 mM) and other nucleoside permeants was low relative to that for NBMPR. The pyrimidine homologue of NBMPR, nitrobenzylthiouridine, also exhibited low affinity for the NBMPR-binding sites. Pretreatment of HeLa cells with p-chloromercuribenzene sulphonate (p-CMBS) or N-ethylmaleimide (NEM) decreased binding of NBMPR to its high-affinity sites and inhibited uridine transport, indicating the presence of thiol groups essential to both processes. NEM, a more penetrable reagent than p-CMBS, inhibited binding and transport at much lower concentrations than the latter compound. Pretreatment of cells with concentrations of p-CMBS that alone had no effect on either NBMPR binding or uridine transport increased the sensitivity of transport to NBMPR inhibition and changed the shape of the NBMPR concentration-effect curve, suggesting synergistic inhibiton of uridine-transport activity by these two agents.
硝基苄硫代肌苷（NBMPR）能与多种动物细胞质膜核苷转运元件上的抑制位点可逆性结合，但亲和力较高（解离常数Kd为0.1 - 1.2 nM）。本研究探讨了HeLa细胞中NBMPR结合与尿苷转运抑制之间的关系。HeLa细胞在悬浮培养和单层培养中尿苷向内转运的Km值约为0.1 mM。相对于NBMPR，转运抑制位点对尿苷（Kd为1.7 mM）、肌苷（Kd为0.4 mM）和其他核苷渗透剂的亲和力较低。NBMPR的嘧啶类似物硝基苄硫代尿苷对NBMPR结合位点也表现出低亲和力。用对氯汞苯磺酸盐（p - CMBS）或N - 乙基马来酰亚胺（NEM）预处理HeLa细胞会降低NBMPR与其高亲和力位点的结合，并抑制尿苷转运，这表明这两个过程都需要巯基的存在。NEM是一种比p - CMBS更易渗透的试剂，在比后者低得多的浓度下就能抑制结合和转运。用单独对NBMPR结合或尿苷转运均无影响的p - CMBS浓度预处理细胞，会增加转运对NBMPR抑制的敏感性，并改变NBMPR浓度 - 效应曲线的形状，表明这两种试剂对尿苷转运活性具有协同抑制作用。
