纽波特绿，一种弱结合细胞锌离子（Zn²⁺）的荧光传感器：与蛋白质组竞争锌离子（Zn²⁺）
Newport Green, a fluorescent sensor of weakly bound cellular Zn(2+): competition with proteome for Zn(2).
作者信息
Karim Mohammad Rezaul, Petering David H
机构信息
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA.
出版信息
Metallomics. 2016 Feb;8(2):201-10. doi: 10.1039/c5mt00167f.
Newport Green (NPG) is a recognized sensor of cellular Zn(2+) that displays fluorescence enhancement upon binding to Zn(2+). Because of its modest affinity for Zn(2+), the extent of its capacity to bind cellular Zn(2+) is unclear. The present study investigated the range of reactivity of NPG(ESTER) with cells, isolated (Zn)-proteome, and model Zn-proteins. The sensor accumulated in pig kidney LLC-PK1 cells and was slowly (>40 min) hydrolyzed to the fluorescent, acid form, NPG(ACID). The powerful, cell permeant Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethane-1,2-diamine (TPEN) failed to quench the growing fluorescence emission, indicating that Zn-NPG(ACID) had not formed and NPG-Zn-protein adduct species probably were not present. Furthermore, NPG(ACID) did not bind to Zn-carbonic anhydrase or Zn-alcohol dehydrogenase, two proteins that form adducts with some other sensors. Strikingly, most of the NPG(ACID) that had been converted from NPG(ESTER) was detected in the extracellular medium not the cells. As a result, after cells were incubated with NPG(ESTER) and then Zn-pyrithione to raise the internal concentration of mobile Zn(2+), Zn-NPG(ACID) was only observed in the external medium. Residual cellular NPG(ACID) was unable to bind extra intracellular Zn(2+) delivered by pyrithione. Proteome isolated from the sonicated cell supernatant was also unreactive with NPG(ACID). Titration of proteome or glutathione with Zn(2+) in the presence of NPG(ACID) revealed that NPG(ACID) only weakly competes for mobile Zn(2+) in the presence of these cellular components. In addition, when proteomic Zn(2+) was released by a nitric oxide donor or N-ethyl-maleimide, little Zn(2+) was detected by NPG(ACID). However, exposure to nitric oxide independently enhanced the fluorescence properties of NPG(ACID). Thus, the biochemical properties of NPG related to cellular Zn(2+) chelation deepen the question of how it functions as a Zn(2+) sensor.
纽波特绿（NPG）是一种公认的细胞锌离子（Zn(2+)）传感器，与Zn(2+)结合后会显示荧光增强。由于其对Zn(2+)的亲和力适中，其结合细胞内Zn(2+)的能力范围尚不清楚。本研究调查了NPG（酯）与细胞、分离的（锌）蛋白质组和模型锌蛋白的反应范围。该传感器在猪肾LLC-PK1细胞中积累，并缓慢（>40分钟）水解为荧光酸性形式NPG（酸）。强大的细胞渗透性Zn(2+)螯合剂N,N,N',N'-四（2-吡啶甲基）-乙烷-1,2-二胺（TPEN）未能淬灭不断增加的荧光发射，这表明未形成Zn-NPG（酸），且可能不存在NPG-Zn-蛋白质加合物。此外，NPG（酸）不与锌碳酸酐酶或锌醇脱氢酶结合，这两种蛋白质会与其他一些传感器形成加合物。引人注目的是，从NPG（酯）转化而来的大部分NPG（酸）是在细胞外培养基中检测到的，而不是在细胞内。因此，在用NPG（酯）孵育细胞，然后用吡啶硫酮锌提高细胞内可移动Zn(2+)的浓度后，仅在外部培养基中观察到Zn-NPG（酸）。残留的细胞内NPG（酸）无法结合由吡啶硫酮提供的细胞内额外的Zn(2+)。从超声处理的细胞上清液中分离的蛋白质组也与NPG（酸）无反应。在NPG（酸）存在的情况下，用Zn(2+)滴定蛋白质组或谷胱甘肽表明，在这些细胞成分存在的情况下，NPG（酸）对可移动Zn(2+)的竞争较弱。此外，当蛋白质组中的Zn(2+)由一氧化氮供体或N-乙基马来酰亚胺释放时，NPG（酸）几乎检测不到Zn(2+)。然而，暴露于一氧化氮会独立增强NPG（酸）的荧光特性。因此，与细胞Zn(2+)螯合相关的NPG的生化特性加深了它如何作为Zn(2+)传感器发挥作用的问题。
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