AJP - Cell Physiology, Vol 269, Issue 3 C739-C749, Copyright © 1995 by American Physiological Society

ARTICLES

S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation

C. M. Padgett and A. R. Whorton
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.

Nitric oxide (NO), produced by vascular endothelial cells, mediates both physiological and pathological responses. Although the molecular targets responsible for NO-mediated endothelial cell injury are not known, one candidate is the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In this study, we investigated the mechanism involved in NO-mediated GAPDH inhibition and found that S-nitrosoglutathione (GSNO) inhibited GAPDH activity in both purified enzyme preparations and endothelial cells. Furthermore, GSNO-mediated GAPDH inhibition occurred by modification of the active site cysteine residue in GAPDH, since increasing concentrations of the substrate, glyceraldehyde-3-phosphate, which interacts with the active site cysteine residue, protected GAPDH from inhibition by GSNO. Although under certain conditions both GSNO and the NO donor, sodium nitroprusside (SNP), led to the covalent NAD(+)-dependent modification of GAPDH, this putative ADP ribosylation was unlikely to be the primary mechanism for inhibition, since the stoichiometry was extremely low, and, in the case of GSNO, inhibition was completely reversed by thiol reagents. Furthermore, GSNO effectively S-nitrosylated GAPDH, and the extent of nitrosylation was linearly correlated with the degree of inhibition such that addition of 1 mole of NO per mole of GAPDH monomer was necessary to inhibit the enzyme. Consistent with this finding, GSNO-mediated GAPDH inhibition was reversible with low-molecular-weight thiols, and the reversal of inhibition correlated with the "denitrosylation" of GAPDH. These results suggest that endothelial GAPDH is a target for NO and that inhibition occurs principally by the reversible S-nitrosylation of the active site cysteine residue in GAPDH.

This article has been cited by other articles:

				S. Li and A. R. Whorton Functional characterization of two S-nitroso-L-cysteine transporters, which mediate movement of NO equivalents into vascular cells Am J Physiol Cell Physiol, April 1, 2007; 292(4): C1263 - C1271. [Abstract] [Full Text] [PDF]

				B. Vergauwen, F. Pauwels, M. Vaneechoutte, and J. J. Van Beeumen Exogenous Glutathione Completes the Defense against Oxidative Stress in Haemophilus influenzae J. Bacteriol., March 1, 2003; 185(5): 1572 - 1581. [Abstract] [Full Text] [PDF]

				A Sola, J Rosello-Catafau, E Gelpi, and G Hotter Fructose-1,6-biphosphate in rat intestinal preconditioning: involvement of nitric oxide Gut, February 1, 2001; 48(2): 168 - 175. [Abstract] [Full Text] [PDF]

				Y. S. Chang, J. A. Yaccino, S. Lakshminarayanan, J. A. Frangos, and J. M. Tarbell Shear-Induced Increase in Hydraulic Conductivity in Endothelial Cells Is Mediated by a Nitric Oxide-Dependent Mechanism Arterioscler. Thromb. Vasc. Biol., January 1, 2000; 20(1): 35 - 42. [Abstract] [Full Text] [PDF]

				S. Mohr, H. Hallak, A. de Boitte, E. G. Lapetina, and B. Brune Nitric Oxide-induced S-Glutathionylation and Inactivation of Glyceraldehyde-3-phosphate Dehydrogenase J. Biol. Chem., April 2, 1999; 274(14): 9427 - 9430. [Abstract] [Full Text] [PDF]

				H. Li, Z. M. Marshall, and A. R. Whorton Stimulation of cystine uptake by nitric oxide: regulation of endothelial cell glutathione levels Am J Physiol Cell Physiol, April 1, 1999; 276(4): C803 - C811. [Abstract] [Full Text] [PDF]

				J. Satriano, S. Ishizuka, D. C. Archer, R. C. Blantz, and C. J. Kelly Regulation of intracellular polyamine biosynthesis and transport by NO and cytokines TNF-alpha and IFN-gamma Am J Physiol Cell Physiol, April 1, 1999; 276(4): C892 - C899. [Abstract] [Full Text] [PDF]

				A. J. Gow, D. G. Buerk, and H. Ischiropoulos A Novel Reaction Mechanism for the Formation of S-Nitrosothiol in Vivo J. Biol. Chem., January 31, 1997; 272(5): 2841 - 2845. [Abstract] [Full Text] [PDF]

				M. Xian, X. Chen, Z. Liu, K. Wang, and P. G. Wang Inhibition of Papain by S-Nitrosothiols. FORMATION OF MIXED DISULFIDES J. Biol. Chem., June 30, 2000; 275(27): 20467 - 20473. [Abstract] [Full Text] [PDF]