S-nitrosoglutathione reversibly inhibits glyceraldehyde-3-
phosphate dehydrogenase by s-nitrosylation.
Padgett, Christine M., and A. Richard Whorton.
Department of Pharmacology, Duke University Medical Center, Durham,
NC 27710
APStracts 2:0150C, 1995.
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 in endothelial cells. Furthermore, GSNO
-mediated GAPDH inhibition occurred by modification of the active site
cysteine residue in GAPDH since increasing concentrations of
substrate, glyceraldehyde-3-phosphate, which interacts with the
active site cysteine residue, protected GAPDH from inhibition by
GSNO. While 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.
Received 26 January 1995; accepted in final form 17 March 1995.
APS Manuscript Number C50-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 28 March 1995.