Purified gamma-glutamyl transpeptidases from tomato exhibit high affinity for glutathione and glutathione S-conjugates.

摘要

gamma-Glutamyl transpeptidases (gammaGTases) are the only enzymes known to hydrolyse the unique N-terminal amide bonds of reduced glutathione (gamma-L-glutamyl-cysteinyl-glycine), oxidized glutathione, and glutathione S-conjugates. Two gammaGTases (I and II) with Km values for glutathione of 110 and 90μM were purified 2977-fold and 2152-fold, respectively, from ripe tomato (Lycopersicon esculentum) pericarp. Both enzymes also hydrolyse dipeptides and other tripeptides with N-terminal, gamma-linkedGlu and the artificial substrates gamma-L-glutamyl-p-nitroanilide and gamma-L-glutamyl(7-amido-4-methylcoumarin). They transfer the glutamyl moiety to water or acceptor amino acids, including L-Met, L-Phe, L-Trp, L-Ala, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid. gammaGTase I and II were released from a wall and membrane fraction of a tomato fruit extract with 1.0 M NaCl, suggesting that they are peripheral membrane proteins. They were further purified by acetone precipitation, DyeMatrex Green A affinity chromatography, and hydrophobic interaction chromatography. The two gammaGTases were resolved by concanavalin A (Con A) affinity chromatography, indicating that they are differentially glycosylated. The native and SDS-denatured forms of both enzymes showed molecular masses of 43 kDa.