Sugimoto M, Kuhlenkamp J, Ookhtens M, Aw T Y, Reeve J, Kaplowitz N
Biochem Pharmacol. 1985 Oct 15;34(20):3643-7. doi: 10.1016/0006-2952(85)90224-2.
A new high performance liquid chromatography (HPLC) method for the separation of gamma-glutamylcysteine (GC) from glutathione (GSH) following derivatization with 1-chloro-2,4-dinitrobenzene (CDNB) was developed using a Vydac C18 column and an acetonitrile-trifluoroacetic acid gradient. When the derivatization of GC, GSH, cysteine, and cysteinylglycine was performed with GSH S-transferase, peak heights for the GC and GSH derivatives were accentuated markedly, suggesting that GC, like GSH, is an enzyme substrate. Subsequently, GC was found to be a substrate for five purified forms of rat hepatic GSH S-transferase. However, the Km for GC was about 6-20 times higher than that for GSH. GSH was a competitive inhibitor of GC-CDNB conjugation, indicating that GC and GSH share the same binding site on the transferase. However, endogenous hepatic GC content in fed rats was only 5.8 +/- 0.1 nmoles/g, three orders of magnitude lower than GSH. Thus, under normal circumstances, GC would not be expected to contribute to detoxification reactions catalyzed by the GSH S-transferases. Its weak interaction with the GSH site of the GSH S-transferases supports the role of the glycine moiety of GSH in enhancing this interaction.
开发了一种新的高效液相色谱（HPLC）方法，该方法使用Vydac C18柱和乙腈 - 三氟乙酸梯度，用于在1 - 氯 - 2,4 - 二硝基苯（CDNB）衍生化后从谷胱甘肽（GSH）中分离γ-谷氨酰半胱氨酸（GC）。当用谷胱甘肽S - 转移酶对GC、GSH、半胱氨酸和半胱氨酰甘氨酸进行衍生化时，GC和GSH衍生物的峰高显著增强，表明GC与GSH一样，是一种酶底物。随后，发现GC是大鼠肝脏中五种纯化形式的谷胱甘肽S - 转移酶的底物。然而，GC的Km值比GSH的Km值高约6 - 20倍。GSH是GC - CDNB共轭反应的竞争性抑制剂，表明GC和GSH在转移酶上共享相同的结合位点。然而，喂食大鼠肝脏中的内源性GC含量仅为5.8±0.1纳摩尔/克，比GSH低三个数量级。因此，在正常情况下，预计GC不会对谷胱甘肽S - 转移酶催化的解毒反应有贡献。它与谷胱甘肽S - 转移酶的GSH位点的弱相互作用支持了GSH的甘氨酸部分在增强这种相互作用中的作用。
