OXYGEN-FREE RADICALS ENHANCE THE NITRIC OXIDE-INDUCED COVALENT NAD(+)-LINKAGE TO NEURONAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
OXYGEN-FREE RADICALS ENHANCE THE NITRIC OXIDE-INDUCED COVALENT NAD(+)-LINKAGE TO NEURONAL GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE
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DOI：
10.1042/bj3090891
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发表时间：
1995-08-01
影响因子：
4.1
通讯作者：
PREMONT, J
中科院分区：
文献类型：
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作者：
MARIN, P;MAUS, M;PREMONT, J
一氧化氮（NO）诱导来自各种组织的甘油醛-3-磷酸脱氢酶（GAPDH）的共价修饰。这种现象以前被解释为自身ADP核糖基化，实际上是NAD（+）与酶的共价结合。在本研究中，我们发现，在[腺苷酸-P-32]NAD（+）存在下，3-吗啉代-悉尼酮亚胺（SIN-1）在刺激来自培养的纹状体神经元的GAPDH共价标记方面比硝普钠（SNP）有效得多（SIN-1和SNP的最大有效浓度分别诱导的NAD（+）标记增加877+/-110和266+/-33%）。两种NO生成化合物的功效差异可能是由于SIN-1额外释放超氧化物，因为超氧化物歧化酶和硝酮5，5 '-二甲基吡咯啉-1-氧化物显著抑制SIN-1诱导的NAD（+）与GAPDH的共价结合。过氧化氢酶和选择性清除剂的羟基自由基，甘露醇和二甲基亚砜，没有改变SIN-1诱导的共价修饰的GAPDH，排除了参与这种现象的羟基自由基。进一步支持了氧自由基在NAD（+）与GAPDH连接中的作用，焦性没食子酸（一种超氧化物发生剂，单独使用无效）增强了SNP诱发的反应。在NO和超氧化物存在下测得的NAD（+）与神经元GAPDH的连接可能涉及巯基，因为暴露于HgCl 2可逆转蛋白质的放射性标记，并可通过用烷化剂N-乙基马来酰亚胺预处理来防止。此外，连苯三酚可增强NO诱导的GAPDH活性抑制，而单独使用连苯三酚无效。总之，本研究表明，超氧阴离子增强NO诱导的共价NAD（+）-连接到GAPDH和酶失活。
Nitric oxide (NO) induces a covalent modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from various tissues. This phenomenon, which has previously been interpreted as an auto-ADP-ribosylation, is in fact a covalent binding of NAD(+) to the enzyme. In the present study, we show that 3-morpholino-sydnonimine (SIN-1) is much more efficient than sodium nitroprusside (SNP) in stimulating the covalent labelling of GAPDH from cultured striatal neurones in the presence of [adenylate-P-32]NAD(+) (877+/-110 and 266+/-33% increase in NAD(+)-labelling induced by maximally effective concentrations of SIN-1 and SNP respectively). The difference in the efficacy of both NO-generating compounds could be due to the additional release of superoxide by SIN-1, since superoxide dismutase and the nitrone 5,5'-dimethyl pyrroline-1-oxide markedly inhibited the SIN-1-induced covalent binding of NAD(+) to GAPDH. Catalase and selective scavengers of hydroxyl radicals, mannitol and dimethyl sulphoxide, did not alter the SIN-1-induced covalent modification of GAPDH, ruling out the involvement of hydroxyl radicals in this phenomenon. Supporting further a role of oxygen free radicals in the NAD(+) linkage to GAPDH, pyrogallol, a superoxide generator, which alone was ineffective, potentiated the SNP-evoked response. The NAD(+) linkage to neuronal GAPDH measured in the presence of NO and superoxide probably involves sulphydryl groups, since the radiolabelling of the protein was reversed by exposure to HgCl2, and prevented by pretreatment with the alkylating agent N-ethylmaleimide. Moreover, the NO-induced inhibition of GAPDH activity was enhanced by pyrogallol, which was ineffective alone. In conclusion, the present study indicates that superoxide anions potentiate NO-induced covalent NAD(+)-linkage to GAPDH and enzyme inactivation.