酿酒酵母NAD(H)激酶Pos5p在细胞抵抗氧化胁迫中的作用

The Role of Saccharomyces cerevisiae NAD(H) Kinase Pos5p on the Defense of Oxidative Stress

摘要:

酿酒酵母线粒体NAD(H)激酶Pos5p显示出重要功能, 其缺失将导致细胞抗氧化性能出现障碍。为了了解Pos5p的抗氧化作用机制及其与调节辅酶NAD(H)和NADP(H)之间的关系, 比较了在不同类型的氧化胁迫试剂作用下, 野生型BY4742、POS5基因缺失体pos5Δ及其回补体pos5Δ/POS5-YEp的生长表型, 同时采用高效液相色谱测定细胞内辅酶含量。结果表明, 在超氧生成试剂甲萘醌(VK3)、过氧化氢(H2O2)和GSH消耗试剂马来酸二乙酯(DEM)存在时, pos5Δ都表现出明显的生长缺陷, 而各抗氧化基因缺失体只在其相应胁迫下表现出生长缺陷。在正常生长条件下, pos5Δ的NADPH含量降低, pos5Δ/POS5-YEp则提高, 表明Pos5p对胞内NADPH的供应有重要作用。在VK3、H2O2和DEM胁迫下, BY4742、pos5Δ及pos5Δ/POS5-YEp的NADP(H)含量均有不同程度的下降, 其中pos5Δ的NADP(H)/NAD(H)比率下降最为严重, 而pos5Δ/POS5-YEp较pos5Δ有明显提高, 这与其氧化胁迫表型相一致。因此, 在细胞面临不同类型的氧化胁迫时, Pos5p都能有效行使其NAD(H)激酶活性, 补充NADP(H)的损耗, 从而对细胞起到抗氧化保护作用。

Abstract:

In Saccharomyces cerevisiae, mitochondria NAD(H) kinase Pos5p has significant function. Absence of POS5 gene leads to antioxidative dysfunction. In order to realize the mechanism of anti-oxidative function of Pos5p and its relation to the conversion of coenzyme NAD(H) to NADP(H), we compared the growth phenotypes of wild-type BY4742, POS5 gene deletion mutant pos5Δ and pos5Δ containing POS5-YEplac195 plasmid (pos5Δ/POS5-YEp) under exposure to different kinds of oxidative reagents, and assayed their intracellular coenzyme concentration by the high performance liquid chromatography (HPLC). The results showed that only the pos5Δ had obvious growth defect under all of the three oxidative reagents, that is, superoxide anion generation reagent menadione (VK3), hydrogen peroxide (H2O2) and GSH consumption reagent diethyl maleate (DEM), while the tested anti-oxidative gene deletion mutants only had growth defect under their corresponding oxidative stress. In the normal growth condition, NADPH content of pos5Δ reduced, while that of pos5Δ/POS5-YEp increased, indicating that Pos5p play an important role on the supply of NADPH. Under the exposure to VK3, H2O2 and DEM, the NADP(H) concentration of BY4742, pos5Δ and pos5Δ/POS5-YEp all reduced, with the ratio of NADP(H) to NAD(H) in pos5Δ decreased most obviously. The research indicated that under different kinds of oxidative stress, Pos5p could exert NAD(H) kinase activity effectively to supply NADP(H) and thus had an important antioxidative defense funtion.