PPD, Biopharmaceutical Department, Middleton WI, USA.
Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, 3210 N. Cramer, Milwaukee, Wisconsin 53201, USA.
Metallomics. 2022 Nov 9;14(11). doi: 10.1093/mtomcs/mfac081.
A recent study investigated the impact of glutathione (GSH) on the transfer of zinc (Zn) from proteome to apo-carbonic anhydrase. Here, we probed the requirement of glutathione for zinc trafficking in LLC-PK1 pig kidney epithelial cells. Depletion of GSH by at least 95% left cells viable and able to divide and synthesize Zn-proteins at the control rate over a 48-h period. Loss of GSH stimulated the accumulation of 2.5x the normal concentration of cellular Zn. According to gel filtration chromatography, differential centrifugal filtration, and spectrofluorimetry with TSQ, the extra Zn was distributed between the proteome and metallothionein (MT). To test the functionality of proteome and/or MT as sources of Zn for the constitution of Zn-proteins, GSH-deficient cells were incubated with CaEDTA to isolate them from their normal source of nutrient Zn. Control cells plus CaEDTA stopped dividing; GSH-depleted cells plus CaEDTA continued to divide at ∼40% the rate of GSH deficient cells. Evidently, proteome and/or MT served as a functional source of Zn for generating Zn-proteins. In vitro insertion of Zn bound to proteome into apo-carbonic anhydrase occurred faster at larger concentrations of Zn bound to proteome. These results support the hypothesis that enhanced transport of Zn into cells drives the conversion of apo-Zn-proteins to Zn-proteins by mass action. Similar results were also obtained with human Jurkat T lymphocyte epithelial cells. This study reveals a powerful new model for studying the chemistry of Zn trafficking, including transport processes, involvement of intermediate binding sites, and constitution of Zn-proteins.
最近的一项研究调查了谷胱甘肽 (GSH) 对锌 (Zn) 从蛋白质组转移到脱辅基碳酸酐酶的影响。在这里，我们探讨了 GSH 在 LLC-PK1 猪肾上皮细胞中对锌运输的需求。通过至少 95%的 GSH 耗竭，细胞仍然存活，并能够在 48 小时内以对照速率分裂和合成 Zn 蛋白。GSH 的丧失刺激了细胞内 Zn 浓度正常水平的 2.5 倍的积累。根据凝胶过滤色谱、差速离心过滤和用 TSQ 的荧光光谱法，额外的 Zn 分布在蛋白质组和金属硫蛋白 (MT) 之间。为了测试蛋白质组和/或 MT 作为 Zn 来源以构成 Zn 蛋白的功能，用 CaEDTA 孵育 GSH 缺乏的细胞，使其与正常的营养 Zn 来源分离。对照细胞加 CaEDTA 停止分裂；GSH 耗尽的细胞加 CaEDTA 继续以 GSH 缺乏的细胞的约 40%的速度分裂。显然，蛋白质组和/或 MT 作为 Zn 的功能性来源，用于生成 Zn 蛋白。在体外，与蛋白质组结合的 Zn 插入到脱辅基碳酸酐酶中发生的速度更快，当与蛋白质组结合的 Zn 浓度更大时。这些结果支持了这样一种假设，即增强的 Zn 向细胞内的运输通过质量作用驱动脱辅基 Zn 蛋白向 Zn 蛋白的转化。用人 Jurkat T 淋巴细胞上皮细胞也得到了类似的结果。这项研究揭示了一个研究 Zn 运输化学的强大新模型，包括运输过程、中间结合位点的参与以及 Zn 蛋白的构成。
