Direct Detection of Glutathione Biosynthesis, Conjugation, Depletion and Recovery in Intact Hepatoma Cells.
Direct Detection of Glutathione Biosynthesis, Conjugation, Depletion and Recovery in Intact Hepatoma Cells.
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DOI：
10.3390/ijms23094733
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发表时间：
2022-04-25
影响因子：
5.6
通讯作者：
中科院分区：
文献类型：
--
作者：
采用核磁共振（NMR）技术监测海藻酸盐包膜的JM-1肝癌细胞中谷胱甘肽的代谢，该细胞灌注含有[3,3 ' -13C2]-胱氨酸的生长培养基。标记培养基灌注20 h后，13C核磁共振谱以13C标记谷胱甘肽的信号为主。一旦13c标记，高强度的谷胱甘肽共振允许在1.2分钟的间隔内获得后续光谱。在这个时间分辨率下，可以监测谷胱甘肽代谢的详细动力学，因为硫醇烷基化剂单溴摩烷（mBBr）被添加到灌注液中。添加大剂量的mBBr会导致13c标记的谷胱甘肽的共振迅速减弱，这是由于mBBr通过烷基化损失了这种代谢物。当谷胱甘肽共振减少时，由于细胞内产生谷胱甘肽-双烷缀合物而产生的新共振被检测到。在mBBr剂量从细胞中清除后，通过13c -谷胱甘肽信号的恢复以及偶联物的冲洗，观察到细胞内谷胱甘肽的补充。这些数据表明，标准核磁共振技术可以直接监测细胞内过程，如谷胱甘肽耗竭，时间分辨率约为< 2分钟。
Nuclear magnetic resonance (NMR) spectroscopy was used to monitor glutathione metabolism in alginate-encapsulated JM-1 hepatoma cells perfused with growth media containing [3,3′-13C2]-cystine. After 20 h of perfusion with labeled medium, the 13C NMR spectrum is dominated by the signal from the 13C-labeled glutathione. Once 13C-labeled, the high intensity of the glutathione resonance allows the acquisition of subsequent spectra in 1.2 min intervals. At this temporal resolution, the detailed kinetics of glutathione metabolism can be monitored as the thiol alkylating agent monobromobimane (mBBr) is added to the perfusate. The addition of a bolus dose of mBBr results in rapid diminution of the resonance for 13C-labeled glutathione due to a loss of this metabolite through alkylation by mBBr. As the glutathione resonance decreases, a new resonance due to the production of intracellular glutathione-bimane conjugate is detectable. After clearance of the mBBr dose from the cells, intracellular glutathione repletion is then observed by a restoration of the 13C-glutathione signal along with wash-out of the conjugate. These data demonstrate that standard NMR techniques can directly monitor intracellular processes such as glutathione depletion with a time resolution of approximately < 2 min.
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影响因子：
3.3
作者：
Keshari, Kayvan R.;Kurhanewicz, John;Jeffries, Rex E.;Wilson, David M.;Dewar, Brian J.;Van Criekinge, Mark;Zierhut, Matthew;Vigneron, Daniel B.;Macdonald, Jeffrey M.
通讯作者：
Macdonald, Jeffrey M.
影响因子：
10.6
作者：
Forman, Henry Jay;Zhang, Hongqiao;Rinna, Alessandra
通讯作者：
Rinna, Alessandra
影响因子：
5.1
作者：
Gamcsik, MP;Millis, KK;Hamill, TG
通讯作者：
Hamill, TG
影响因子：
3.8
作者：
Glicklis, R;Merchuk, JC;Cohen, S
通讯作者：
Cohen, S
影响因子：
3.8
作者：
Gamcsik, MP;Forder, JR;McGovern, KA
通讯作者：
McGovern, KA