title: Preferential proteolysis and activation of oxidatively modified liver microsomal glutathione S-transferase of rat.
abstract: Proteolytic activation of oxidatively modified microsomal GSH S-transferase (GSTm) was investigated. When GSTm was incubated with diamide -diazenedicarboxylic acid bis(N,N-dimethylamide)- or hydrogen peroxide in the presence or absence of glutathione, a protein-gluathione mixed-disulfide and a dimer of the enzyme were formed with a concomitant increase in transferase activity. Although control GSTm was activated 3.4-fold by 3 micrograms/ml of trypsin, the monomeric form of the transferase in which the sulfhydryl group was modified by mixed-disulfide bond formation or by covalent binding with N-ethylmaleimide was further stimulated by lower concentrations of trypsin than that used in the control. In contrast, no activation of the dimeric transferase was observed with any concentration of trypsin. In immunoblot analysis, a proteolytic product (fragment A) from the dimer transferase was detected after treatment of oxidant-modified microsomes with low concentrations of trypsin, whereas the fragment (fragment B) from the unmodified-monomeric enzyme was observed by high concentrations of trypsin. These results show that oxidatively modified GSTm is sensitive to proteolytic attack by trypsin and that only monomeric transferase is further activated by limited proteolysis.
authors.affiliation: Laboratory of Physiology and Pharmacology, University of the Ryukyus, Okinawa, Japan.
authors.forename: M
authors.initials: M
authors.lastname: Shimoji
authors.forename: Y
authors.initials: Y
authors.lastname: Aniya
authors.forename: M W
authors.initials: MW
authors.lastname: Anders
mesh_terms: D000818:Animals; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D051381:Rats; D017207:Rats, Sprague-Dawley; D014357:Trypsin / Q000494:pharmacology
chemical_list: D005982:Glutathione Transferase; D014357:Trypsin