Spectrophotometric Method for Determining Glyoxalase 1 Activity in Cerebral Cavernous Malformation (CCM) Disease.

Glyoxalase 1 (Glo1) is a glutathione (GSH)-dependent enzyme that catalyzes the isomerization of the hemithioacetal formed non-enzymatically from methylglyoxal (MG) and GSH to S-D-lactoylglutathione (SLG). The activity of Glo1 is measured spectrophotometrically by following the increase of absorbance at 240 nm and 25 °C, attributable to the formation of SLG. The hemithioacetal is preformed by incubation of 2 mM MG and 1 mM GSH in 0.1 M sodium phosphate buffer (PBS) pH 7.2, at 25 °C for 10 min. The cell extract is then added, and the A is monitored over 5-min incubation against correction for blank. Glo1 activity is given in units per mg of protein where one unit activity is defined as 1 μmole of SLG produced per min under assay conditions. Here, we describe measurement of Glo1 activity in established cellular models of cerebral cavernous malformation (CCM) disease, including KRIT1-knockout mouse embryonic fibroblast (MEF) and KRIT1-silenced human brain microvascular endothelial (hBMEC) cells.