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The role of glutathione (GSH) in eukaryotic cells is well known. The biosynthesis of this γ-glutamine tripeptide is well studied. However, other γ-glutamyl peptides were found in various sources, and the pathways of their formation were not always clear. The aim of the present study was to determine whether Saccharomyces cerevisiae can produce γ-glutamyl tripeptides other than GSH and to identify the pathways associated with the formation of these peptides. The tripeptide γ-Glu-Val-Gly (γ-EVG) was used as a model. Wild-type yeast cells were shown to produce this peptide during cultivation in minimal synthetic medium. Two different biosynthetic pathways for this peptide were identified. The first pathway consisted of two steps. In the first step, γ-Glu-Val (γ-EV) was produced from glutamate and valine by the glutamate-cysteine ligase (GCL) Gsh1p or by the transfer of the γ-glutamyl group from GSH to valine by the γ-glutamyltransferase (GGT) Ecm38p or by the (Dug2p-Dug3p) 2 complex. In the next step, γ-EV was combined with glycine by the glutathione synthetase (GS) Gsh2p. The second pathway consisted of transfer of the γ-glutamyl residue from GSH to the dipeptide Val-Gly (VG). This reaction was carried out mainly by the (Dug2p-Dug3p) 2 complex, whereas the GGT Ecm38p did not participate in this reaction. The contribution of each of these two pathways to the intracellular pool of γ-EVG was dependent on cultivation conditions. In this work, we also found that Dug1p, previously identified as a Cys-Gly dipeptidase, played an essential role in the hydrolysis of the dipeptide VG in yeast cells. It was also demonstrated that γ-EV and γ-EVG could be effectively imported from the medium and that γ-EVG was imported by Opt1p, known to be a GSH importer. Our results demonstrated that γ-glutamyl peptides, particularly γ-EVG, are produced in yeast as products of several physiologically important reactions and are therefore natural components of yeast cells.

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A method to determine cysteine content in food materials using 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxidiazole (ABD-F) was compared to a method using N-acridinyl maleimide (NAM). Cysteine in samples was derivatized with two fluorogenic reagents and analyzed using HPLC. The cysteine derivative with ABD-F gave a single peak while the cysteine derivative with NAM produced three peaks in standard. The cysteine content in model samples determined with ABD-F was nearly identical to the content determined with NAM. However, when the cysteine content of six types of food materials was measured, four samples were identical in each method, but two samples produced different results because the separation of cysteine derivatives with NAM was influenced by other compounds in the food samples. Since the ABD-F method gave the better separation, it can be useful for the determination of cysteine content in food materials.Keywords: 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxidiazole (ABD-F), N-acridinylmaleimide (NAM), cysteine *To whom correspondence should be addressed. E-mail: hiroaki_nishiuchi@ajinomoto.com IntroductionL-Cysteine, one of the 20 natural amino acids, is an important sulfur-containing compound because it is the only source of sulfur for cell metabolism in most organisms. Cysteine is indispensable for protein folding, protein assembly, and protein stability through disulfide bond formation with the reactive thiol residue. In addition to this metabolic importance, cysteine also plays an important industrial role. Cysteine has been used for industrial applications such as pharmaceuticals, cosmetics, bakery, and flavorings, resulting in consumption of approximately 4000 tons/y globally (Wada et al., 2006). For example, cysteine was reported to be effective as a reducing agent in production of French bread, crackers, and cookies (Grandvoinnet et al., 1979). In the beverage industry, cysteine was effective in preventing browning of fruit juice, such as grape juice or pear juice, during concentration (Skalski and Sistrunk, 1974; Montgomery, 1983.) In addition, cysteine prevented the development of off-flavor in stored orange juice . In flavor chemistry, cysteine is an important source of sulfur in a variety of aromas (Strakenmann et al., 2008). Thus, cysteine will continue to play an important role in the food industry. Therefore, a reliable method to determine the cysteine content in food materials is necessary.Several methods have been developed to determine cysteine content. Previously, mercaptide-forming reactions using metal ions were used. However, more recent methods use fluorescent reagents to react with thiol residues. For example, NAM, a maleimide reagent, provides blue fluorescence by reaction with thiol compounds and has been used for analysis of thiol compounds such as cysteine or glutathione (Nara et al., 1978;Takahashi et al., 1979;Meguro et al., 1996). In addition, NAM has been used to determine cysteine content in commercial yeast extracts (Kuroda et al., 1997). In contrast, ABD-F, a reagent with a halo...