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 240nm and 25°C, attributable to the formation of SLG. The hemithioacetal is preformed by incubation of 2mM MG and 1mM GSH in 0.1M sodium phosphate buffer (PBS) pH7.2, at 25°C for 10min. The cell extract is then added, and the A240 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 mumole 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.
Spectrophotometric method for determining glyoxalase 1 activity in cerebral cavernous malformation (CCM) disease
Antognelli C.
;Talesa V. N.;
2020
Abstract
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 240nm and 25°C, attributable to the formation of SLG. The hemithioacetal is preformed by incubation of 2mM MG and 1mM GSH in 0.1M sodium phosphate buffer (PBS) pH7.2, at 25°C for 10min. The cell extract is then added, and the A240 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 mumole 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.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
