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Updated: Jun 13, 2026
Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases
Published on: October 10, 2020
Activation of rat liver microsomal glutathione transferase by hepsin
Shinji Nakama1, Natsuki Oshiro, Yoko Aniya
1Laboratory of Molecular Genetics and Pharmacology, School of Health Sciences, Faculty of Medicine, University of the Ryukyus, USA.
Abstract:
Rat liver microsomal glutathione transferase (MGST1) is activated by limited proteolysis. Recently we purified a protease, hepsin, from rat liver microsomes that activates MGST1. In the present study the mechanism of MGST1 activation by hepsin was investigated. When MGST1 and hepsin were incubated at room temperature, MGST1 activity was markedly increased and the increase was decreased to the control level by further incubation with disulfide bond reducing agent dithiothreitol. MGST1 dimer was detected by electrophoresis after treatment of MGST1 with hepsin, instead of proteolytic product. MGST1 dimer formation accompanied by an increase in MGST1 activity was observed even in the presence of the protease inhibitor benzamidine. Furthermore, prolonged incubation of both enzymes caused the formation of MGST1 dimer and its proteolytic product. These results clearly show that the protease hepsin stimulates disulfide-linked MGST1 dimer formation resulting in activation of MGST1 and preferential degradation of MGST1 dimer. Since hepsin contains disulfide bonds in the scavenger receptor cysteine-rich (SRCR) domain, it was suggested that the SRCR domain interacts with MGST1 leading to thiol/disulfide exchange between the two enzymes followed by disulfide-linked MGST1 dimer formation.
Insights
Rat liver microsomal glutathione transferase (MGST1) is activated by hepsin through disulfide-linked dimer formation, not proteolysis. This interaction enhances MGST1 activity before its degradation.
Area of Science:
- Biochemistry
- Enzymology
- Molecular Biology
Background:
- Microsomal glutathione transferase 1 (MGST1) activity is regulated by limited proteolysis.
- Hepsin, a rat liver microsomal protease, was previously identified as an activator of MGST1.
Purpose of the Study:
- To investigate the mechanism by which hepsin activates MGST1.
- To elucidate the role of disulfide bonds and protein interactions in MGST1 activation.
Main Methods:
- Incubation of MGST1 and hepsin at room temperature.
- Treatment with dithiothreitol (a reducing agent) to assess disulfide bond involvement.
- Electrophoresis to detect MGST1 dimer formation.
- Use of the protease inhibitor benzamidine to differentiate proteolysis from other activation mechanisms.
Main Results:
- Hepsin treatment markedly increased MGST1 activity, which was reversed by dithiothreitol.
- Electrophoresis revealed MGST1 dimer formation, not a proteolytic product, upon hepsin treatment.
- MGST1 dimer formation and increased activity occurred even with a protease inhibitor.
- Prolonged incubation led to both MGST1 dimer formation and subsequent proteolytic degradation of the dimer.
Conclusions:
- Hepsin activates MGST1 by stimulating the formation of disulfide-linked MGST1 dimers.
- This disulfide-linked dimer formation leads to increased MGST1 activity.
- Hepsin subsequently facilitates the degradation of the activated MGST1 dimer.
- The scavenger receptor cysteine-rich (SRCR) domain of hepsin likely mediates this interaction via thiol/disulfide exchange.
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