Evidence for pH dependent Zn2+ influx in K562 erythroleukemia cells:: Studies using ZnAF-2F fluorescence and 65Zn2+ uptake
Evidence for pH dependent Zn2+ influx in K562 erythroleukemia cells:: Studies using ZnAF-2F fluorescence and 65Zn2+ uptake
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DOI：
10.1016/j.abb.2005.08.013
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发表时间：
2005-10-15
影响因子：
3.9
通讯作者：
Kikuchi, K
中科院分区：
文献类型：
--
作者：
Colvin, RA;Fontaine, CP;Kikuchi, K
利用ZnAF-2F（一种Zn2+特异性荧光团）和Zn-65(2+)，我们确定了稳态条件下转运体介导的Zn2+内流速率（可能由SLC39A1基因产物介导，蛋白名称为hZIP1），并研究了细胞外酸化的影响。将K562红细胞置于含Zn2+的缓冲液（1-60 μ m）中，Zn-65（2+）积累的初始速率反映了ZnAF-2F检测到的细胞内游离Zn2+浓度的明显上升。因此，新运输的Zn2+与ZnAF-2F感知的胞内自由Zn2+池平衡。约30 min后，细胞内游离Zn2+升高，形成新的稳态。Zn2+内流的K-m值为11 μ m， V-max值为0.203 nmol/mg/s。细胞外酸化抑制Zn-65（2+）摄取和ZnAF-2F荧光变化（测试范围：pH 8-6， IC50 = pH 6.34）。质子效应的IC50接近组氨酸的pK(a)，表明SLC39A1中存在的保守组氨酸残基在Zn2+内流中起关键作用，并参与pH效应。(c) 2005爱思唯尔公司版权所有。
Using both ZnAF-2F (a Zn2+ specific fluorophore) and Zn-65(2+), we determined the rate of transporter mediated Zn2+ influx (presumably mediated by the SLC39A1 gene product, protein name hZIP1) under steady state conditions and studied the effects of extracellular acidification. When K562 erythroleukemia cells were placed in Zn2+ containing buffers (1-60 mu m), the initial rate of Zn-65(2+) accumulation mirrored the apparent rise in free intracellular Zn2+ concentrations sensed by ZnAF-2F. Therefore, newly transported Zn2+ equilibrated with the free intracellular Zn2+ pool sensed by ZnAF-2F. A new steady state with elevated free intracellular Zn2+ was established after about 30 min. An estimate of 11 mu m for the K-m and 0.203 nmol/mg/s for the V-max were obtained for Zn2+ influx. Zn-65(2+) uptake and ZnAF-2F fluorescent changes were inhibited by extracellular acidification (range tested: pH 8-6, IC50 = pH 6.34). The IC50 for proton effects was close to the pK(a) for histidine, suggesting conserved histidine residues present in SLC39A1 play a critical role in Zn2+ influx and are involved in the pH effect. (c) 2005 Elsevier Inc. All rights reserved.