Dagnino L, Bennett L L, Paterson A R
Cancer Research Group (McEachern Laboratory), University of Alberta, Edmonton, Canada.
J Biol Chem. 1991 Apr 5;266(10):6312-7.
Two equilibrative (facilitated diffusion) nucleoside transport processes and a concentrative Na(+)-dependent co-transport process contribute to zero-trans inward fluxes of nucleosides in L1210 mouse leukemia cells. Na(+)-linked inward adenosine fluxes in L1210/AM cells (a clone deficient in adenosine, deoxyadenosine, and deoxycytidine kinase activities) were measured as initial rates of [3H]adenosine influx in medium containing Na+ salts and 10 microM dipyridamole. The Na(+)-linked transporter distinguished between the D- and L-enantiomers of adenosine, the latter being a virtual nonpermeant in the initial-rate assay. Adenine arabinoside, inosine, 2'-deoxyadenosine and 2'-deoxyadenosine derivatives with halogen atoms at the purine C-2 position were recognized as substrates of the Na(+)-linked system because of their inhibition of adenosine (10 microM) fluxes under the condition of Na(+)-dependence with IC50 values ranging between 25 and 183 microM; uridine, deoxycytidine, and cytosine arabinoside (each at 400 microM) inhibited adenosine fluxes by 10-40%. Inward Na(+)-linked adenosine fluxes were saturable with respect to extracellular adenosine and Na+ concentrations [( Na+]o); Km and Vmax values for adenosine influx were 9.4 +/- 2.6 microM and 1.67 +/- 0.2 pmol/microliter cell water/s when [Na+]o was 100 mM. The stoichiometry of Na+:adenosine co-transport, determined by Hill analysis of the dependence of adenosine fluxes on [Na+]o, was 1:1. The thiol-reactive agents, N-ethylmaleimide (NEM), showdomycin and p-chloromercuriphenylsulphonate (pCMPS), inhibited Na(+)-linked adenosine fluxes with IC50 values of 40, 10, and 2 microM, respectively. This inhibition was partially reversed by the presence of adenosine in incubation media containing pCMPS, but not NEM. Thiol groups accessible to pCMPS may be involved in substrate recognition by the transporter and in the permeation step.
两种平衡型（易化扩散）核苷转运过程和一种依赖钠离子的同向转运过程共同促成了L1210小鼠白血病细胞中核苷的零转运内向通量。通过测量在含有钠盐和10微摩尔双嘧达莫的培养基中[3H]腺苷流入的初始速率，来测定L1210/AM细胞（一种腺苷、脱氧腺苷和脱氧胞苷激酶活性缺陷的克隆细胞）中与钠离子相关的内向腺苷通量。与钠离子相关的转运体能够区分腺苷的D型和L型对映体，在初始速率测定中，后者实际上是不可通透的。阿糖腺苷、肌苷、2'-脱氧腺苷以及在嘌呤C-2位带有卤素原子的2'-脱氧腺苷衍生物，由于它们在钠离子依赖条件下对腺苷（10微摩尔）通量的抑制作用（IC50值在25至183微摩尔之间），被认为是与钠离子相关系统的底物；尿苷、脱氧胞苷和阿糖胞苷（各400微摩尔）使腺苷通量降低了10 - 40%。内向的与钠离子相关的腺苷通量相对于细胞外腺苷和钠离子浓度[（Na +）o]是可饱和的；当[（Na +）o]为100毫摩尔时，腺苷流入的Km和Vmax值分别为9.4±2.6微摩尔和1.67±0.2皮摩尔/微升细胞水/秒。通过对腺苷通量对[（Na +）o]的依赖性进行希尔分析确定，钠离子与腺苷同向转运的化学计量比为1:1。硫醇反应剂N - 乙基马来酰亚胺（NEM）、多氧霉素和对氯汞苯磺酸盐（pCMPS）分别以40、10和2微摩尔的IC50值抑制与钠离子相关的腺苷通量。在含有pCMPS但不含NEM的孵育培养基中，腺苷的存在可部分逆转这种抑制作用。pCMPS可及的硫醇基团可能参与了转运体对底物的识别以及通透步骤。
