Measuring picomolar intracellular exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor
Measuring picomolar intracellular exchangeable zinc in PC-12 cells using a ratiometric fluorescence biosensor
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DOI：
10.1021/cb500043a
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发表时间：
2006-01-01
影响因子：
4
通讯作者：
Fierke, Carol A.
中科院分区：
文献类型：
--
作者：
Bozym, Rebecca A.;Thompson, Richard B.;Fierke, Carol A.
锌在生物学中发挥着生理和病理作用，使其越来越受到人们的关注。迄今为止，已在补充或富含锌的细胞类型（例如海马神经元）中测量了细胞内游离锌。在这里，我们使用基于碳酸酐酶 (CA) 的激发比率荧光生物传感器对普通静息细胞培养系 (PC-12) 中的细胞内可交换锌进行定量成像。人 CA 11 对锌的 K-d 为 4 pM，并且不受毫摩尔钙或镁离子的干扰。基于CA的生物传感器很容易通过一种新方法引入细胞中：将转录反式激活因子（TAT）衍生的细胞穿透肽与CA分子融合并将其添加到细胞中。我们的结果表明，细胞质和细胞核中的静息浓度约为 5-10 μM。有趣的是，四（2-吡啶甲基）乙二胺（TPEN）-Zn 复合物和 TPEN 对该细胞系均具有凋亡作用。
Zinc plays both physiological and pathological roles in biology, making it of increasing interest. To date, intracellular free zinc has been measured in cell types supplemented with or enriched in zinc, such as hippocampal neurons. Here we quantitatively image intracellular exchangeable zinc in an ordinary resting cell culture line (PC-12), using an excitation ratiometric fluorescent biosensor based on carbonic anhydrase (CA). Human CA 11 has a K-d of 4 pM for zinc and suffers no interference from millimolar calcium or magnesium ions. The CA-based biosensor was readily introduced into the cell by a novel approach: fusing a transactivator of transcription (TAT)-derived cell penetrating peptide to the CA molecule and adding it to the cells. Our results indicate that the resting concentration is approximately 5-10 mu M in cytoplasm and nucleus. Interestingly, the tetrakis(2-pyridylmethyl)ethylenediamine (TPEN)-Zn complex and TPEN are both apoptogenic for this cell line.