Newport Green, a fluorescent sensor of weakly bound cellular Zn(2+): competition with proteome for Zn(2).
Newport Green, a fluorescent sensor of weakly bound cellular Zn(2+): competition with proteome for Zn(2).
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Newport Green，一种弱结合细胞 Zn(2) 的荧光传感器：与蛋白质组竞争 Zn(2)。
DOI：
10.1039/c5mt00167f
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Petering,DavidH
中科院分区：
文献类型：
--
作者：
Karim,MohammadRezaul;Petering,DavidH
Newport Green (NPG) is a recognized sensor of cellular Zn2+that displays fluorescence enhancement upon binding to Zn2+. Because of its modest affinity for Zn2+, the extent of its capacity to bind cellular Zn2+is unclear. The present study investigated the range of reactivity of NPGESTERwith cells, isolated (Zn)-proteome, and model Zn-proteins. The sensor accumulated in pig kidney LLC-PK1cells and was slowly (>40 min) hydrolyzed to the fluorescent, acid form, NPGACID. The powerful, cell permeant Zn2+chelator,N,N,N′,N′-tetrakis(2-pyridylmethyl)-ethane-1,2-diamine (TPEN) failed to quench the growing fluorescence emission, indicating that Zn–NPGACIDhad not formed and NPG–Zn-protein adduct species probably were not present. Furthermore, NPGACIDdid not bind to Zn-carbonic anhydrase or Zn-alcohol dehydrogenase, two proteins that form adducts with some other sensors. Strikingly, most of the NPGACIDthat had been converted from NPGESTERwas detected in the extracellular medium not the cells. As a result, after cells were incubated with NPGESTERand then Zn-pyrithione to raise the internal concentration of mobile Zn2+, Zn–NPGACIDwas only observed in the external medium. Residual cellular NPGACIDwas unable to bind extra intracellular Zn2+delivered by pyrithione. Proteome isolated from the sonicated cell supernatant was also unreactive with NPGACID. Titration of proteome or glutathione with Zn2+in the presence of NPGACIDrevealed that NPGACIDonly weakly competes for mobile Zn2+in the presence of these cellular components. In addition, when proteomic Zn2+was released by a nitric oxide donor orN-ethyl-maleimide, little Zn2+was detected by NPGACID. However, exposure to nitric oxide independently enhanced the fluorescence properties of NPGACID. Thus, the biochemical properties of NPG related to cellular Zn2+chelation deepen the question of how it functions as a Zn2+sensor.
