PREFERENTIAL PROTEOLYSIS AND ACTIVATION OF OXIDATIVELY MODIFIED LIVERMICROSOMAL GLUTATHIONE-S-TRANSFERASE OF RAT

Proteolytic activation of oxidatively modified microsomal GSH S-transf erase (GSTm) was investigated. When GSTm was incubated with diamide [d iazenedicarboxylic acid bis(N,N-dimethylamide)] or hydrogen peroxide i n the presence or absence of glutathione, a protein-glutathione mixed- disulfide and a dimer of the enzyme were formed with a concomitant inc rease in transferase activity. Although control GSTm was activated 3.4 -fold by 3 mu g/ml of trypsin, the monomeric form of the transferase i n which the sulfhydryl group was modified by mixed-disuIfide bond form ation or by covalent binding with N-ethylmaleimide was further stimula ted by lower concentrations of trypsin than that used in the control, In contrast, no activation of the dimeric transferase was observed wit h any concentration of trypsin, In immunoblot analysis, a proteolytic product (fragment A) from the dimer transferase was detected after tre atment of oxidant-modified microsomes with low concentrations of tryps in, whereas the fragment (fragment B) from the unmodified-monomeric en zyme was observed by high concentrations of trypsin. These results sho w that oxidatively modified GSTm is sensitive to proteolytic attack by trypsin and that only monomeric transferase is further activated by l imited proteolysis.