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, Howe ver, it was not possible, using this assay, to detect any enzyme activity i n situ, in Saccharomyces cerevisiae permeabilized cells. Glyoxalase II acti vity 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 substrat e 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) m g(-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 wit h permeabilized cell concentration. gamma-Glutamyl transferase (EC 2.3.2.2) , which also accepts S-D-lactoylglutathione as substrate and hence could in terfere with glyoxalase II assays, was found to be absent in Saccharomyces cerevisiae permeabilized cells. (C) 1999 Academic Press.