Padgett C M, Whorton A R
Department of Pharmacology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Am J Physiol. 1995 Sep;269(3 Pt 1):C739-49. doi: 10.1152/ajpcell.1995.269.3.C739.
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.
血管内皮细胞产生的一氧化氮（NO）介导生理和病理反应。虽然尚不清楚负责NO介导的内皮细胞损伤的分子靶点，但其中一个候选靶点是糖酵解酶甘油醛-3-磷酸脱氢酶（GAPDH）。在本研究中，我们调查了NO介导的GAPDH抑制所涉及的机制，发现S-亚硝基谷胱甘肽（GSNO）在纯化的酶制剂和内皮细胞中均抑制GAPDH活性。此外，GSNO介导的GAPDH抑制是通过修饰GAPDH中的活性位点半胱氨酸残基而发生的，因为与活性位点半胱氨酸残基相互作用的底物甘油醛-3-磷酸浓度增加可保护GAPDH免受GSNO的抑制。虽然在某些条件下，GSNO和NO供体硝普钠（SNP）都会导致GAPDH发生共价的NAD(+)依赖性修饰，但这种假定的ADP核糖基化不太可能是抑制的主要机制，因为化学计量极低，而且就GSNO而言，抑制作用可被硫醇试剂完全逆转。此外，GSNO有效地使GAPDH发生S-亚硝基化，亚硝基化程度与抑制程度呈线性相关，以至于每摩尔GAPDH单体添加1摩尔NO才能抑制该酶。与这一发现一致，GSNO介导的GAPDH抑制作用可被低分子量硫醇逆转，抑制作用的逆转与GAPDH的“脱亚硝基化”相关。这些结果表明，内皮GAPDH是NO的靶点，抑制作用主要通过GAPDH活性位点半胱氨酸残基的可逆S-亚硝基化而发生。
