Specific S-thiolation of a 30-kDa cytosolic protein from rat liver under oxidative stress.
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.
Rokutan
K
K
Institut für Physiologische Chemie I, Universität Düsseldorf, Federal Republic of Germany.
Thomas
J A
JA
Sies
H
H
D000818:Animals; D002458:Cell Fractionation; D003600:Cytosol / Q000378:metabolism; D003958:Diamide / Q000494:pharmacology; D004220:Disulfides / Q000096:biosynthesis*; D005978:Glutathione / Q000031:analogs & derivatives / Q000494:pharmacology; D019803:Glutathione Disulfide; D007525:Isoelectric Focusing; D008099:Liver / Q000378:metabolism*; D008297:Male; D010545:Peroxides / Q000494:pharmacology; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013438:Sulfhydryl Compounds / Q000494:pharmacology*; D013997:Time Factors; D020122:tert-Butylhydroperoxide
D004220:Disulfides; D010545:Peroxides; D011506:Proteins; D013438:Sulfhydryl Compounds; D003958:Diamide; D020122:tert-Butylhydroperoxide; D005978:Glutathione; D019803:Glutathione Disulfide