Glyoxalase I is a novel nitric-oxide-responsive protein

To clarify the molecular mechanisms of nitric oxide (NO) signalling, we exa mined the NO-responsive proteins in cultured human endothelial cells by two -dimensional (2D) PAGE. Levels of two proteins [NO-responsive proteins (NOR Ps)] with different pi values responded to NO donors. One NORP (pI 5.2) app eared in response to NO, whereas another (pI 5.0) disappeared. These protei ns were identified as a native form and a modified form of human glyoxalase I (Glox I; EC 4.4.1.5) by peptide mapping, microsequencing and correlation between the activity and the isoelectric shift. Glox I lost activity in re sponse to NO, and all NO donors tested inhibited its activity in a dose-dep endent manner. Activity and normal electrophoretic mobility were restored b y dithiothreitol and by the removal of sources of NO from the culture mediu m. Glox I was selectively inactivated by NO; compounds that induce oxidativ e stress (H2O2, paraquat and arsenite) failed to inhibit this enzyme. Our r esults suggest that NO oxidatively modifies Glox I and reversibly inhibits the enzyme's activity. The inactivation of Glox I by NO was more effective than that of glyceraldehyde-3-phosphate dehydrogenase (G3PDH), another NO-s ensitive enzyme. Thus Glox I seems to be a novel NO-responsive protein that is more sensitive to NO than G3PDH.