McDonald L J, Moss J
Laboratory of Cellular Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD 20892.
Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6238-41. doi: 10.1073/pnas.90.13.6238.
Nitric oxide-stimulated modification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by [adenylate-32P]NAD has been interpreted in recent reports as ADP-ribosylation. Incubations of GAPDH with the NO-releasing agent sodium nitroprusside (SNP) and NAD resulted, however, in essentially equal incorporation of radiolabel from the adenine, phosphate, and nicotinamide moieties to the extent of approximately 0.02 mol of NAD.mol of GAPDH-1. Modification of GAPDH by free adenosine 5'-diphosphoribose (ADP-ribose) was only 10% of that by NAD. Exposure of GAPDH modified by NAD in the presence of SNP to HgCl2, which acts at thiol linkages, released two products. Both contained nicotinamide and adenylate but did not cochromatograph with NAD. GAPDH activity was inhibited by SNP in a dose-dependent manner in the presence of NAD. When inhibition was 80%, with 1 mM SNP and 1 mM dithiothreitol, covalent modification with NAD was < 2%. This result is consistent with the conclusion that inhibition of GAPDH activity by SNP in the presence of NAD is due primarily to active-site nitrosylation, as reported by other workers, and is not due to the minor modification with NAD. These results demonstrate that NO-stimulated modification of GAPDH with NAD is not ADP-ribosylation as previously reported but rather is covalent binding of NAD through a NO-dependent thiol intermediate, possibly providing an example of an unexpected, altered reactivity of a nitrosylated protein.
近期报告将一氧化氮刺激下甘油醛-3-磷酸脱氢酶（GAPDH）被[腺苷酸-32P]NAD修饰解释为ADP-核糖基化。然而，将GAPDH与一氧化氮释放剂硝普钠（SNP）和NAD一起孵育时，腺嘌呤、磷酸和烟酰胺部分的放射性标记掺入量基本相等，达到约0.02摩尔NAD·摩尔GAPDH-1的程度。游离腺苷5'-二磷酸核糖（ADP-核糖）对GAPDH的修饰仅为NAD的10%。在SNP存在下用NAD修饰的GAPDH暴露于作用于硫醇键的HgCl2时，释放出两种产物。两者都含有烟酰胺和腺苷酸，但与NAD的色谱行为不一致。在NAD存在下，SNP以剂量依赖的方式抑制GAPDH活性。当抑制率达到80%时，加入1 mM SNP和1 mM二硫苏糖醇，NAD的共价修饰<2%。这一结果与其他研究人员报道的结论一致，即在NAD存在下SNP对GAPDH活性的抑制主要是由于活性位点亚硝基化，而不是由于与NAD的少量修饰。这些结果表明，一氧化氮刺激下GAPDH与NAD的修饰并非如先前报道的那样是ADP-核糖基化，而是通过一氧化氮依赖的硫醇中间体与NAD的共价结合，这可能提供了一个亚硝基化蛋白意外改变反应性的例子。
