McDonald B, Reep B, Lapetina E G, Molina y Vedia L
Wellcome Research Laboratories, Research Triangle Park, NC 27709.
Proc Natl Acad Sci U S A. 1993 Dec 1;90(23):11122-6. doi: 10.1073/pnas.90.23.11122.
Nitric oxide (NO) or NO-generating compounds like sodium nitroprusside (SNP) increase cellular levels of cGMP and produce S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase [GAPDH; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12]. In search of a reagent that could discriminate between these two effects, we used the sesquiterpene antibiotic koningic acid, which binds to GAPDH at the Cys-149 of the active site. Koningic acid inhibited basal and sodium nitroprusside-stimulated NAD-dependent covalent modification of purified rabbit muscle GAPDH in a dose-dependent manner. Furthermore, we tested the effect of koningic acid on human platelets. Approximately 90% of GAPDH is present in the cytosol of human platelets, and the exposure of platelet cytosol to koningic acid inhibited GAPDH activity, while the soluble guanylyl cyclase (basal and sodium nitroprusside-stimulated) activity remained unaltered. Pretreatment of intact platelets with koningic acid slowed the rate of aggregation induced by a submaximal concentration of thrombin. In addition, the antibiotic also inhibited the cGMP increases triggered by SNP, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosyndomidine (SIN-1) but failed to prevent an increase in cGMP caused by nitrosylated albumin. Under the same conditions, koningic acid also inhibited basal and SNP- SNAP-, and SIN-1-stimulated NAD-dependent modification of GAPDH and its enzymatic activity. These results suggest that the mechanism of delivery of NO from SNP, SNAP, and SIN-1 to platelets may require the active form of GAPDH. When NO is delivered by nitrosylated albumin, active GAPDH was not necessary.
一氧化氮（NO）或诸如硝普钠（SNP）等能产生NO的化合物会增加细胞内cGMP的水平，并使甘油醛-3-磷酸脱氢酶[GAPDH；D-甘油醛-3-磷酸：NAD+氧化还原酶（磷酸化），EC 1.2.1.12]发生S-亚硝基化。为了寻找一种能够区分这两种效应的试剂，我们使用了倍半萜抗生素 koningic 酸，它在活性位点的Cys-149处与GAPDH结合。Koningic酸以剂量依赖的方式抑制纯化的兔肌肉GAPDH的基础和硝普钠刺激的NAD依赖性共价修饰。此外，我们测试了koningic酸对人血小板的影响。大约90%的GAPDH存在于人血小板的细胞质中，血小板细胞质与koningic酸接触会抑制GAPDH活性，而可溶性鸟苷酸环化酶（基础和硝普钠刺激的）活性保持不变。用koningic酸预处理完整血小板会减缓由次最大浓度凝血酶诱导的聚集速率。此外，该抗生素还抑制了由SNP、S-亚硝基-N-乙酰青霉胺（SNAP）和3-吗啉代辛多明（SIN-1）引发的cGMP增加，但未能阻止亚硝基化白蛋白引起的cGMP增加。在相同条件下，koningic酸还抑制基础和SNP-、SNAP-以及SIN-1刺激的GAPDH的NAD依赖性修饰及其酶活性。这些结果表明，从SNP、SNAP和SIN-1向血小板递送NO的机制可能需要活性形式的GAPDH。当通过亚硝基化白蛋白递送NO时，活性GAPDH不是必需的。
