Glyoxalase II in Saccharomyces cerevisiae:: In situ kinetics 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 of S-D-lactoylglutathione, However, it was not possible, using this assay, to detect any enzyme activity in situ, in Saccharomyces cerevisiae permeabilized 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 II in situ using S-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) x 10(-2) mu mol min(-1) mg(-1) in permeabilized cells and (3.90 +/- 0.04) x 10-2 pmol min(1) mg(-1) in cell-free extracts. Kinetic parameters were K-m 0.36 +/- 0.09 mM and V (7.65 +/- 0.59) x 10(-4) mM min-l for permeabilized cells and K-m 0.15 +/- 0.10 mM and V (7.23 +/- 1.04) x 10(-4) mM min(-1) for cell-free extracts, D-Lactate concentration was also determined and increased in a linear way with permeabilized cell concentration. gamma-Glutamyl transferase (EC 2.3.2.2), which also accepts S-D-lactoylglutathione as substrate and hence could interfere with glyoxalase II assays, was found to be absent in Saccharomyces cerevisiae permeabilized cells. (C) 1999 Academic Press.