Formation of 5-Oxoproline from Glutathione in Erythrocytes by the γ-Glutamyltranspeptidase-Cyclotransferase Pathway

Abstract

-y-Glutamyltranspeptidase activity was demonstrated in the membrane fraction of rabbit erythrocytes. The activity observed (with glutathione and various amino-acid acceptors) was similar in magnitude to that of the -y-glutamylcyclotransferase and -y-glutamylcysteine synthetase activities found in the soluble fraction of the cell. No transpeptidase activity was observed with either -y-glutamyl p-nitroanilide or oxidized glutathione in contrast to the rabbit-kidney enzyme for which these compounds and glutathione serve as substrates. Erythrocyte suspensions and hemolysates formed 5-oxoproline (pyroglutamate; pyrrolidone carboxylate); the rate of 5-oxoproline formation from glutathione by hemolysates was increased by addition of methionine. The findings indicate that 5-oxoproline is an end-product of glutathione metabolism in erythrocytes, and that 5-oxoproline passes out of the erythrocyte and is metabolized in other tissues. The observed rate of 5-oxoproline formation is consistent with the conclusion that the -y-glutamyltranspeptidase-cyclotransferase pathway, together with the synthesis of glutathione from glycine, cysteine, and glutamate, account for a large fraction of the observed amino-acid turnover of erythrocyte glutathione.Although mature erythrocytes do not synthesize protein, they are significantly active in the synthesis of the trilel)tide, glutathione. The functions of erythrocyte glutathione are not fully understood, but there is evidence that glutathione plays an important role in erythrocyte metabolism and in the protection of hemoglobin against oxidation (1, 2). Much attention has been given to the reversible oxidation of glutathione, and it has been suggested that glutathione may function in the regulation of the hexose monol)hosp)hate pathway (3,4). Erythrocyte glutathione exhibits an amino-acid turnover that is relatively slow (several days) as compared, e.g., to that of the liver (5). The enzymes that catalyze the synthesis of glutathione have been found in and purified from erythrocytes (6-8), but little information is available about the metabolic fate of erythrocyte glutathione. It has been suggested that the observed turnover of glutathione in circulating erythrocytes is due to oxidation of glutathione to the corresponding disulfide followed by active transport of the oxidized form of glutathione out of the erythrocyte (9); thus, according to this interl)retation glutathione is not degraded to its constituent amino acids within the erythrocyte.In the present work, we have examined the possibility that erythrocyte glutathione is degraded by a pcathway similar to that which takes place in several other cell types, i.e., enzymatic transl)el)tidation of glutathione with amino acids to form y-glutamyl amino acids and cysteinylglycine (10).Cysteinylglycine is split by a peptidase to its constituent amino acids, and y-glutamyl amino acids are converted by the 293 action of 'y-glutamylcyclotransferase (11)(12)(13)(14) to the corresponding amino acids and 5-oxoproline*. Thus, glutathione ...

Emerging Regulatory Paradigms in Glutathione Metabolism

“…Although GGCT was first purified nearly 40 years ago (Board et al, 1978; Orlowski et al, 1969; Palekar et al, 1974), the gene encoding this enzyme has only recently been identified (Oakley et al, 2008). It shares sequence and structural similarity to γ-glutamylamine cyclotransferase, which degrades γ-glutamyl-ɛ-lysine liberated from proteins covalently crosslinked by transglutaminases (Oakley et al, 2010).…”

“…Initial studies by Meister and co-workers indicated that GGCT purified from rat liver has relatively broad substrate specificity, consistent with the relatively non-specific transfer of a γ-glutamyl moiety to acceptor substrates by GGT. These observations led to the proposal of a GGT/GGCT pathway for import of amino acids, as a component function of the γ-glutamyl cycle (Orlowski et al, 1969; Palekar et al, 1974). However, GGCT involvement in amino acid salvage has not been directly demonstrated using systems in which enzyme expression has been experimentally altered.…”

Biochemical heterogeneity in glutathione synthetase deficiency.

“…The effluent was lyophilized and then the material was dissolved in distilled water and fractionated on Dowex-1 (formate) according to Wilk and Orlowski (20). The oxoproline-containing fraction was lyophilized and repurified by paper chromatography (21). The 5-oxoproline was then converted to L-glutamic acid and assayed with glutamic dehydrogenase (EC 1.4.1.3) (5).…”

Synthesis and GGCT Inhibitory Activity of <i>N</i>-Glutaryl-L-alanine Analogues

“…These results show that carboxylic acid units in the alanine and glutaryl terminals are important for the inhibition. Ring-containing substrates 8 to 10 were only minimally effective, showing at best only 20% inhibition (entries 7,8,9). Compounds with mono and dimethyl groups substituents at the 2-position, 11 (49%) and 13 (52%), showed 2-7 times greater inhibitory activity than those located at the 4-position, 12 (23%) and 14 (7%) in entries 10-13.…”