Differential mechanisms of inhibition of glyceraldehyde-3-phosphate dehydrogenase by S-nitrosothiols and NO in cellular and cell-free conditions. Am J Physiol Heart Circ Physiol 2010 Oct;299(4):H1212-9
Date
08/03/2010Pubmed ID
20675567Pubmed Central ID
PMC2957357DOI
10.1152/ajpheart.00472.2010Scopus ID
2-s2.0-77958046248 (requires institutional sign-in at Scopus site) 29 CitationsAbstract
S-nitrosothiols are nitric oxide (NO)-derived molecules found in biological systems. They have been variously discussed as both NO reservoirs and as major actors in NO-dependent, but cGMP-independent, signal transduction. Although S-nitrosation of specific cysteine residues has been suggested to represent a novel redox-based signaling mechanism, the exact mechanisms of S-nitrosothiol formation under (patho)physiological conditions and the determinants of signaling specificity have not yet been established. Here we examined the sensitivity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) to inhibition by S-nitrosocysteine (CysNO) and NO both intracellularly and in isolation. Bovine aortic endothelial cells (BAECs) and purified GAPDH preparations were treated with CysNO or NO, and enzymatic activity was monitored. Intracellular GAPDH was irreversibly inhibited upon CysNO administration, whereas treatment with NO resulted in a DTT-reversible inhibition of the enzyme. Purified GAPDH was inhibited by both CysNO and NO, but the inhibition pattern was diametrically opposite to that observed in the cells; CysNO-dependent inhibition was reversed with DTT, whereas NO-dependent inhibition was not. In the presence of GSH, NO inhibited purified GAPDH in a DTT-reversible way. Our data suggest that in response to CysNO treatment, cellular GAPDH undergoes S-nitrosation, which results in an irreversible inhibition of the enzyme under turnover conditions. In contrast, NO inhibits the enzyme via oxidative mechanisms that do not involve S-nitrosation and are reversible. In summary, our data show that GAPDH is a target for CysNO- and NO-dependent inhibition; however, these two agents inhibit the enzyme via different mechanisms both inside the cell and in isolation. Additionally, the differences observed between the cellular system and purified protein strongly imply that the intracellular environment dictates the mechanism of inhibition.
Author List
Broniowska KA, Hogg NAuthor
Neil Hogg PhD Associate Dean, Professor in the Biophysics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
AnimalsAorta
Cattle
Cells, Cultured
Cysteine
Endothelium, Vascular
Glyceraldehyde-3-Phosphate Dehydrogenases
Models, Animal
Nitric Oxide
S-Nitrosothiols
Signal Transduction
