THE USE OF MONOCHLOROBIMANE TO DETERMINE HEPATIC GSH LEVELS AND SYNTHESIS
THE USE OF MONOCHLOROBIMANE TO DETERMINE HEPATIC GSH LEVELS AND SYNTHESIS
复制标题
DOI：
10.1016/0003-2697(90)90183-a
复制
发表时间：
1990-11-01
影响因子：
2.9
通讯作者：
KAPLOWITZ, N
中科院分区：
文献类型：
--
作者：
FERNANDEZCHECA, JC;KAPLOWITZ, N
我们用一氯二亚胺（mBcCl）与GSH的特异性反应来分析肝GSH。mBCl本身无荧光，在GSH S-转移酶（GST）催化的反应中与GSH形成稳定的荧光加合物。当肝细胞在Krebs-Henseleit缓冲液中用mBCl（100 μ M）标记时，随时间记录的荧光信号与GSH浓度成正比。预装载染料的肝细胞的HPLC分析表明，GSH是唯一的巯基标记。将该技术应用于新鲜分离的含有不同水平GSH的完整肝细胞，发现用本方法（mBCl）测定的GSH水平与标准酶循环法测定的GSH水平之间有密切的相关性，两种方法测定的细胞溶质和线粒体GSH库也有相似的一致性。荧光GSH-bimane加合物一旦在细胞内形成，就不会从细胞中释放出来。此外，我们已经应用这种技术来直接确定GSH在无细胞条件下和在细胞悬浮液中的合成速率，通过监测荧光加合物的增加，当mBCl存在于过量的孵育。
We have used the specific reaction of monochlorobimane (mBcCl) with GSH to analyze hepatic GSH. mBCl, itself nonfluorescent, forms a stable, fluorescent adduct with GSH in a reaction catalyzed by the GSH S-transferases (GST). When hepatocytes were labeled with mBCl (100 .mu.M) in Krebs-Henseleit buffer, the fluorescent signal recorded over time was directly proportional to the concentration of GSH. The HPLC analyses of hepatocytes that were preloaded with the dye indicated that GSH was the only thiol labeled. When the technique was applied to freshly isolated intact hepatocytes that contained different levels of GSH, a close correlation between the levels of GSH measured by the present method (mBCl) and th standard enzymatic recycling method was found. A similar agreement for the cytosolic and mitochondrial pools of GSH determined by the two methods was established. The fluorescent GSH-bimane adduct, once formed within the cell, was not released from the cell. In addition, we have applied this technique to determine directly the rate of synthesis of GSH in both cell-free conditions and in cell suspensions by monitoring the increase in fluorescent adduct when mBCl is present in excess in the incubation.