STUDIES ON THE REVERSIBILITY OF PROTEIN S-THIOLATION IN HUMAN ENDOTHELIAL-CELLS

Exposure of human umbilical vein endothelial cells to oxidants, such a s hydrogen peroxide and diamide, has been shown to induce protein-spec ific S-thiolation of cellular proteins. In this study we have now iden tified glutathione (reduced form) as the major low molecular-weight th iol that is bound to protein substrates in human umbilical vein endoth elial (HUVE) cells during oxidative stress and investigated the dose- and time-response relationship of diamide- and hydrogen peroxide-induc ed S-thiolation of HUVE cell protein. Intact HUVE cells are able to ra pidly reduce S-thiolated proteins with almost quantitative reappearanc e of reduced glutathione in the cells and protection from acute, lytic cytotoxicity. Additionally, studies were performed with detergent-sol ubilized cell extracts to determine the nature of the reductants opera ting in HUVE cells to maintain protein thiol homeostasis. The results clearly show the involvement of NADH- and NADPH-dependent systems. The se data suggest that the reversible S-thiolation of proteins in these human endothelial cells may represent a significant cellular antioxida nt and regulatory mechanism during oxidative stress. (C) 1994 Academic Press, Inc.