Gamma-glutamylcysteine: a substrate for glutathione S-transferases.
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.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004151:Dipeptides / Q000032:analysis* / Q000378:metabolism; D005978:Glutathione / Q000032:analysis* / Q000378:metabolism; D005982:Glutathione Transferase / Q000378:metabolism*; D066298:In Vitro Techniques; D007700:Kinetics; D008099:Liver / Q000201:enzymology / Q000378:metabolism*; D008297:Male; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity
D004151:Dipeptides; D005982:Glutathione Transferase; D005978:Glutathione; C017341:gamma-glutamylcysteine