MOLECULAR MECHANISM OF ACTIN S-THIOLATION IN GASTRIC MUCOSAL DEFENSE.

氧化应激可诱导蛋白质与低分子量硫醇形成混合二硫化物，该过程称为S-硫醇化。有人认为，这一过程可能会调节氧化应激下的细胞代谢事件。我们用^ S对细胞内谷胱甘肽池进行放射性标记后，通过凝胶电泳和放射自显影分析了培养的豚鼠胃粘膜细胞中特定可溶性蛋白的S-巯基化<35>。过氧化氢（0.2 mM）或二酰胺（0.2 mM）产生快速和可逆的S-巯基化的一组不同的蛋白质与分子量为42，30，29，28，和22千道尔顿（kD）。细胞中S-硫醇化的程度与细胞内还原型谷胱甘肽的损失和与试剂孵育期间发生的蛋白结合型谷胱甘肽的增加成比例。过氧化氢引起突出的30 kD蛋白质的S-硫醇化1分钟内。二酰胺开始突出的42 kD蛋白质的S-硫醇化，和修改持续超过20分钟的研究期间。通过免疫印迹分析和肌动球蛋白沉淀鉴定该蛋白为肌动蛋白。荧光显微镜显示，联酰胺引起的正常应力纤维的消失，并伴随着增加肌动蛋白聚合与收缩的细胞。这些形态学变化是完全可逆的，并且时间过程与肌动蛋白的S-巯基化比脱巯基化大致相同。相反，通过与丁硫氨酸-（R，S）-亚砜亚胺孵育耗尽谷胱甘肽的细胞，细胞从培养板上脱离。肌动蛋白的S-巯基化可以帮助保护胃粘膜细胞免受氧化应激下微丝的不可逆组织。

Oxidative stress can induce the formation of protein mixed disulfides with low-molecular-weight thiols, a process termed S-thiolation. It has been suggested that this process might modulate cellular metabolic events under oxidative stress. We analyzed S-thiolation of specific soluble proteins in cultured gastric mucosal cells from guinea pigs by gel electrophoresis and autoradiography after radiolabeling of the intracellular glutathione pool with^<35>S. Hydrogen peroxide (0.2 mM) or diamide (0.2 mM) produced rapid and reversible S-thiolation of a distinct group of proteins with molecular masses of 42, 30, 29, 28, and 22 kilodalton (kD). The extent of S-thiolation in the cells was proportional to the loss of intracellular reduced glutathione and the increase in protein-bound glutathione that occurred during incubation with the agents. Hydrogen peroxide caused prominent S-thiolation of the 30 kD protein within 1 min. Diamide initiated prominent S-thiolation of the 42 kD protein, and the modification persisted over the 20 min study period. This protein was identified as actin by immunoblot analysis and actomyosin precipitation. Fluorescent microscopy revealed that diamide caused a disappearance of normal stress fiber and a concomitant increase in actin polymerization in associated with contraction of the cells. These morphological changes were completely reversible, and the time course was approximately the same as that of the S-thiolation, than dethiolation of actin. In contrast, with cells depleted of glutathione by incubation with buthionine-(R,S)-sulfoximine, the cells detached from the culture plates. S-thiolation of actin could help protect the gastric mucosal cells against irreversible organization of microfilaments under oxidative stress.