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 μM tert‐butyl hydroperoxide, 50 μM diamide [1,1‐azobis(N,N′‐dimethylformamide)] or 20 μM 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. Copyright © 1989, Wiley Blackwell. All rights reserved