Glyoxalase II inSaccharomyces cerevisiae: In SituKinetics Using the 5,5′-Dithiobis(2-nitrobenzoic Acid) Assay

The determination of glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) activity is usually accomplished by monitoring the decrease of absorbance at 240 nm due to the hydrolysis ofS-d-lactoylglutathione. However, it was not possible, using this assay, to detect any enzyme activityin situ,inSaccharomyces cerevisiaepermeabilized cells. Glyoxalase II activity was then determined by following the formation of GSH at 412 nm using 5,5′-dithiobis(2-nitrobenzoic acid). Using this method we characterized the kinetics of glyoxalase IIin situusingS-d-lactoylglutathione as substrate and compared the results with those obtained for cell-free extracts. The specific activity was found to be (4.08 ± 0.12) × 10−2μmol min−1mg−1in permeabilized cells and (3.90 ± 0.04) × 10−2μmol min1mg−1in cell-free extracts. Kinetic parameters wereKm0.36 ± 0.09 mM andV(7.65 ± 0.59) × 10−4mM min−1for permeabilized cells andKm0.15 ± 0.10 mM andV(7.23 ± 1.04) × 10−4mM min−1for cell-free extracts.d-Lactate concentration was also determined and increased in a linear way with permeabilized cell concentration. γ-Glutamyl transferase (EC 2.3.2.2), which also acceptsS-d-lactoylglutathione as substrate and hence could interfere with glyoxalase II assays, was found to be absent inSaccharomyces cerevisiaepermeabilized cells.