Fourrat Latifa, Iddar Abdelghani, Valverde Federico, Serrano Aurelio, Soukri Abdelaziz
Laboratoire de Physiologie et Génétique moléculaire, Département de Biologie, Faculté des Sciences Aïn-Chock, Université Hassan-II, Km 8 route d'El Jadida, B.P. 5366 Mâarif, Casablanca, Morocco.
J Eukaryot Microbiol. 2007 Jul-Aug;54(4):338-46. doi: 10.1111/j.1550-7408.2007.00275.x.
Previous reports showed that hydrogen peroxide and the NO-generating reagent sodium nitroprusside (SNP)-modulated enzymatic activity of animal glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). These modifications are suggested to have a physiological regulatory role. To gain further insight into this regulatory process the model ciliated protozoan Tetrahymena pyriformis was chosen. Both reagents inhibited growth of T. pyriformis cultures and produced a specific increase of GAPDH protein but only NO seemed to reduce GAPDH activity in cell-free extracts. Both specific activity and pI were found to be altered in the in vivo NO-treated purified enzyme, but no effect was detected by the in vivo H(2)O(2) treatment. Analytical chromatofocusing showed a single basic isoform (pI 8.8) in enzyme preparations from control and H(2)O(2)-treated cells. In contrast to this, three more acidic isoforms (pIs, 8.6, 8.0 and 7.3) were resolved in purified fractions from SNP-treated cells, suggesting post-translational modification of the enzyme by NO. Nevertheless, a decrease of GAPDH activity by H(2)O(2) and NO, mainly due to a decrease in its V(max) without apparent change in substrate affinity, was observed in vitro in the whole enzyme population. The increase of GAPDH protein level found in vivo suggests a cell response in order to compensate for the inhibitory effect on activity observed in the purified enzyme. This is the first report of NO- and H(2)O(2)-dependent effects on GAPDH of T. pyriformis, and identifies this key protein of central carbon metabolism as a physiological target of oxidative and nitrosative stress in this ciliated protozoan.
先前的报告表明，过氧化氢和产生一氧化氮的试剂硝普钠（SNP）可调节动物甘油醛-3-磷酸脱氢酶（GAPDH，EC 1.2.1.12）的酶活性。这些修饰被认为具有生理调节作用。为了进一步深入了解这一调节过程，选择了模式纤毛虫梨形四膜虫。两种试剂均抑制梨形四膜虫培养物的生长，并使GAPDH蛋白特异性增加，但只有一氧化氮似乎能降低无细胞提取物中的GAPDH活性。在体内经一氧化氮处理的纯化酶中，发现比活性和等电点均发生了改变，但体内过氧化氢处理未检测到任何影响。分析性色谱聚焦显示，来自对照细胞和过氧化氢处理细胞的酶制剂中存在单一的碱性同工型（pI 8.8）。与此相反，在经SNP处理细胞纯化的组分中解析出另外三种酸性同工型（pI分别为8.6、8.0和7.3），表明一氧化氮对该酶进行了翻译后修饰。然而，在体外整个酶群体中观察到，过氧化氢和一氧化氮均使GAPDH活性降低，这主要是由于其Vmax降低，而底物亲和力无明显变化。体内发现的GAPDH蛋白水平升高表明细胞做出了反应，以补偿在纯化酶中观察到的对活性的抑制作用。这是关于一氧化氮和过氧化氢对梨形四膜虫GAPDH依赖性影响的首次报告，并确定这种中心碳代谢的关键蛋白是这种纤毛虫中氧化应激和亚硝化应激的生理靶点。
