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

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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.