CELLULAR-RECOVERY OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE-ACTIVITY AND THIOL STATUS AFTER EXPOSURE TO HYDROPEROXIDES
CELLULAR-RECOVERY OF GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE-ACTIVITY AND THIOL STATUS AFTER EXPOSURE TO HYDROPEROXIDES
复制标题
DOI：
10.1016/0003-9861(90)90028-w
复制
发表时间：
1990-01-01
影响因子：
3.9
通讯作者：
REED, DJ
中科院分区：
文献类型：
--
作者：
BRODIE, AE;REED, DJ
在脊椎动物细胞中，巯基依赖性酶甘油醛-3-磷酸脱氢酶（GPD）的活性被细胞内巯基二硫氧化还原状态的变化所调节。人肺癌细胞（A549）用1-120 mM H2O2、1-120 mM过氧化氢t-丁基、1-6 mM乙酸或0.1-10 mM n -乙基马来酰亚胺孵育5分钟。通过硫醇试剂碘乙酸与GPD的结合来测量，还原蛋白硫醇的损失和GPD酶活性的损失以剂量依赖的方式发生。氧化处理后，将细胞在生理盐水中孵育30分钟或用20毫米二硫苏糖醇（DTT）部分逆转GPD的这两种变化。在不向培养基中添加硫醇的情况下，或者用含有2mm半胱氨酸、半胱氨酸、半胱胺或谷胱甘肽（GSH）的超声细胞混合物孵育，可以观察到GPD活性的酶促恢复；GSSG没有效果。用丁硫氨酸亚砜（BSO）处理细胞以不同剂量降低细胞GSH，导致GPD活性对H2O2失活的敏感性随剂量增加而增加，并降低细胞随后恢复的能力。GPD对另一种肺癌细胞系（A427）以及来自人和大鼠的正常肺组织的氧化处理也有类似的反应。这些发现表明，细胞硫醇氧化还原状态在确定GPD酶活性方面可能是重要的。
The activity of the thiol-dependent enzyme glyceraldehyde-3-phosphate dehydrogenase (GPD), in vertebrate cells, was modulated by a change in the intracellular thiol:disulfide redox status. Human lung carcinoma cells (A549) were incubated with 1-120 mM H2O2, 1-120 mM t-butyl hydroperoxide, 1-6 mM ethacrynic acid, or 0.1-10 mM N-ethylmaleimide for 5 min. Loss of reduced protein thiols, as measured by binding of the thiol reagent iodoacetic acid to GPD, and loss of GPD enzymatic activity occurred in a dose-dependent manner. Incubation of the cells, following oxidative treatment, in saline for 30 min or with 20 mM dithiothreitol (DTT) partially reversed both changes in GPD. The enzymatic recovery of GPD activity was observed either without addition of thiols to the medium or by incubation of a sonicated cell mixture with 2 mM cysteine, cystine, cysteamine, or glutathione (GSH); GSSG had no effect. Treatment of cells with buthionine sulfoximine (BSO) to decrease cellular GSH by varying amounts caused a dose-related increase in sensitivity of GPD activity to inactivation by H2O2 and decreased cellular ability for subsequent recovery. GPD responded in a similar fashion with oxidative treatment of another lung carcinoma cell line (A427) as well as normal lung tissue from human and rat. These findings indicate that the cellular thiol redox status can be important in determining GPD enzymatic activity.