Specific S-thiolation of a 30-kDa cytosolic protein from rat liver under oxidative stress

Abstract

Thin-gel isoelectric focusing (IEF) is a simple and sensitive method of quantifying S-thiolation of individual proteins (protein mixed-disulfide formation). IEF of rat liver cytosol identified one major protein (pI 7.0) which underwent S-thiolation with glutathione disulfide to produce two acidic bands with pIs 6.4 and 6.1. The S-thiolated forms of the protein were purified by preparative isoelectric focusing. An apparent molecular mass of 30 kDa was determined by SDS/polyacrylamide gel electrophoresis. The 30-kDa protein amounted to 7 +/- 2% of the total cytosolic protein on IEF. The most abundant soluble protein of freshly isolated hepatocytes, with an identical isoelectric point to the liver 30-kDa protein, was modified in a similar manner in response to oxidative stress induced by model compounds. Addition of 50 microM tert-butyl hydroperoxide, 50 microM diamide [1,1-azobis(N,N'-dimethylformamide)] or 20 microM menadione (2-methyl-1,4-naphthoquinone) initiated the S-thiolation within less than 2 min in the hepatocytes. These compounds, at the concentrations employed, did not result in cell death. Menadione produced slowly progressive S-thiolation of the protein, while tert-butyl hydroperoxide or diamide produced rapid S-thiolation that decreased quickly after 2 min.