Glutathione (L-gamma-glutamyl-L-cysteinylglycine) is physiologically synthesized through two ATP-dependent reactions catalyzed by gamma-glutamylcysteine synthase and glutathione synthase. The present study was designed to produce glutathione without the aid of ATP by using glutathione-degrading enzymes, gamma-glutamyl transpeptidase and aminopeptidase M, in reverse: intrinsically the former enzyme catalyzes the cleavage of glutathione to give L-cysteinylglycine and a gamma-glutamyl moiety and the latter hydrolyzes the peptide linkage of L-cysteinylglycine. Both enzymes were simultaneously displayed on proteoliposomes, which were reconstituted from bovine kidney brush border membranes by a cholate dialysis method. The kinetic analysis using artificial substrates, L-gamma-glutamyl-p-nitroanilide for gamma-glutamyl transpeptidase and L-leucine-p-nitroanilide for aminopeptidase M, revealed that the proteoliposome reconstitution significantly increased the enzyme activities: for both the enzymes the maximum reaction rates were increased and Michaelis constants with the respective substrates were decreased. When the proteoliposomes were incubated with the amino acids glycine, L-cysteine, and L-glutamate (or L-glutamine) at 37degreesC, a new product was determined on HPLC analyses using ODS and cation-exchange columns, coinciding in retention time with authentic glutathione. This product was identified to be glutathione by LC-MS and H-1-NMR, after being purified by gel filtration using Sephadex G10 and HSKgel Toyopearl HW-40F in succession. When the incubation mixture contained acivicin and bestatin, specific inhibitors for gamma-glutamyl transpeptidase and aminopeptidase M,, respectively, glutathione was not produced at all. These results indicated that glutathione was produced by two-step reversible reactions of aminopeptidase ML and gamma-glutamyl transpeptidase from its constituent amino acids. The equilibrium glutathione concentration obtained with L-glutamine as a glutamyl donor substrate was about 3.5 times higher than that obtained with L-glutamate. The maximum pH for the glutathione production was 7.0-7.5, reflecting pH dependence of the activities of the enzymes. (C) 2004 Society of Chemical Industry.