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Preferential proteolysis and activation of oxidatively modified liver microsomal glutathione S-transferase of rat
M Shimoji1, Y Aniya, M W Anders
1Laboratory of Physiology and Pharmacology, University of the Ryukyus, Okinawa, Japan.
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.
Insights
Oxidative modification of microsomal glutathione S-transferase (GSTm) leads to increased activity and sensitivity to proteolysis. Limited proteolysis by trypsin further activates monomeric GSTm, but not dimeric forms.
Area of Science:
- Biochemistry
- Enzymology
- Oxidative Stress
Background:
- Microsomal glutathione S-transferase (GSTm) plays a crucial role in cellular defense against oxidative damage.
- Oxidative modification can alter enzyme structure and function, impacting its catalytic activity.
- Understanding the regulation of GSTm activity under oxidative stress is vital for cellular protection mechanisms.
Purpose of the Study:
- To investigate the proteolytic activation of oxidatively modified microsomal glutathione S-transferase (GSTm).
- To determine how oxidative modifications affect GSTm's sensitivity to trypsin and its subsequent activation.
- To elucidate the differential effects of proteolysis on monomeric versus dimeric forms of GSTm.
Main Methods:
- Incubation of GSTm with oxidants (diamide, hydrogen peroxide) and glutathione.
- Assay of transferase activity following oxidative modification and/or proteolysis.
- Limited proteolysis using varying concentrations of trypsin.
- Immunoblot analysis to detect proteolytic fragments.
Main Results:
- Oxidative modification of GSTm resulted in the formation of a protein-glutathione mixed-disulfide and enzyme dimers, with increased transferase activity.
- Oxidatively modified monomeric GSTm showed enhanced activation by lower concentrations of trypsin compared to control GSTm.
- Dimeric GSTm, whether oxidatively modified or not, was not activated by trypsin.
- Immunoblot analysis revealed distinct trypsin digestion patterns for monomeric and dimeric GSTm, indicating structural differences.
Conclusions:
- Oxidatively modified GSTm is susceptible to proteolytic cleavage by trypsin.
- Limited proteolysis selectively activates the monomeric form of oxidatively modified GSTm.
- The study highlights a novel regulatory mechanism for GSTm activity involving oxidative stress and proteolysis.