Glutathione peroxidase activities from rat liver

Area of Science:

• Biochemistry
• Enzymology

Background:

• Rat liver possesses two enzymes exhibiting glutathione peroxidase activity using cumene hydroperoxide as a substrate.
• One enzyme is selenium-dependent (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9), while the other is independent of dietary selenium.

Purpose of the Study:

• To characterize the selenium-independent glutathione peroxidase from rat liver.
• To investigate its kinetic properties, molecular weight, and potential identity with glutathione S-transferase.

Main Methods:

• Enzyme activity assays using cumene hydroperoxide and 1-chloro-2,4-dinitrobenzene.
• Gel filtration and SDS-polyacrylamide gel electrophoresis for molecular weight determination.
• Kinetic analysis using double reciprocal plots and substrate inhibition studies.

Main Results:

• The selenium-independent enzyme has an estimated molecular weight of 35,000 and subunit molecular weight of 17,000.
• Kinetic analysis suggested a sequential reaction mechanism with Km values of 0.20 mM for glutathione and 0.57 mM for cumene hydroperoxide.
• The enzyme was inhibited by N-ethylmaleimide and cyanide, but not iodoacetic acid. It also catalyzed glutathione conjugation to 1-chloro-2,4-dinitrobenzene.

Conclusions:

• The selenium-independent glutathione peroxidase exhibits characteristics similar to glutathione S-transferase.
• These findings suggest that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver may be attributed to the same enzyme.