Identification of an abundant S-thiolated rat liver protein as carbonic anhydrase III; characterization of S-thiolation and dethiolation reactions.
An S-thiolated 30-kDa protein has been purified from rat liver by two steps of ion-exchange chromatography. This monomeric protein has two "reactive" sulfhydryls that can be S-thiolated by glutathione (form a mixed disulfide with glutathione) in intact liver. The protein has been identified as carbonic anhydrase III by sequence analysis of tryptic peptides from the pure protein. The two "reactive" sulfhydryls on this protein can produce three different S-thiolated forms of the protein that can be separated by isoelectric focusing. Using this technique it was possible to study the S-thiolation and dethiolation reactions of the pure protein. The reduced form of this protein was S-thiolated both by thiol-disulfide exchange with glutathione disulfide and by oxyradical-initiated S-thiolation with reduced glutathione. The S-thiolation rate of this 30-kDa protein was somewhat slower than that of glycogen phosphorylase b by both S-thiolation mechanisms. The S-thiolated form of this protein was poorly dethiolated (i.e., reduced) by glutathione, cysteine, cysteamine, or coenzyme A alone. Enzymatic catalysis by two different enzymes (glutaredoxin and thioredoxin-like) greatly enhanced the dethiolation rate. These experiments suggest that carbonic anhydrase III is a major participant in the liver response to oxidative stress, and that the protein may be S-thiolated by two different non-enzymatic mechanisms and dethiolated by enzymatic reactions in intact cells. Thus, the S-thiolation/dethiolation of carbonic anhydrase III resembles glycogen phosphorylase and not creatine kinase.
Chai
Y C
YC
Department of Biochemistry, Iowa State University, Ames 50011.
Jung
C H
CH
Lii
C K
CK
Ashraf
S S
SS
Hendrich
S
S
Wolf
B
B
Sies
H
H
Thomas
J A
JA
D000595:Amino Acid Sequence; D000818:Animals; D002256:Carbonic Anhydrases / Q000032:analysis*; D002852:Chromatography, Ion Exchange; D003065:Coenzyme A / Q000378:metabolism; D004220:Disulfides / Q000378:metabolism; D005609:Free Radicals; D054477:Glutaredoxins; D005978:Glutathione / Q000378:metabolism; D007525:Isoelectric Focusing; D008099:Liver / Q000201:enzymology*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D013379:Substrate Specificity; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism
D004220:Disulfides; D005609:Free Radicals; C516010:Glrx2 protein, rat; D054477:Glutaredoxins; D011506:Proteins; D013438:Sulfhydryl Compounds; D013879:Thioredoxins; D010088:Oxidoreductases; D002256:Carbonic Anhydrases; D005978:Glutathione; D003065:Coenzyme A