The activity of glutathione reductase under liver pathologies and enzyme purification by ion-exchange chromatography for the catalytic properties study
Abstract
This work is devoted to the analysis of glutathione reductase activity in patients with non-alcoholic
steatohepatitis, developing on a background of type 2 diabetes, in patients with drug-induced hepatitis, at an
experimental toxic hepatitis in animals, as well as the isolation of the enzyme from rat liver by ion-exchange
and gel-chromatography and its regulatory properties study. The blood serum of people, treated in hospital,
and healthy persons with normal total and biochemical blood tests was used in the course of the experiment.
Besides, the object of the study were control white male laboratory rats and animals with toxic hepatitis
modeled by oral administration of carbon tetrachloride. Glutathione reductase activity was determined spectrophotometrically
at 340 nm. Reaction rate was assessed by optical density drop as a result of NADPH oxidation.
Protein content was determined by the Lowry method. Purification of the enzyme from experimental
animals liver included several steps: homogenization of liver sample in a porcelain mortar in 4-fold volume of cooled isolation medium followed by fractionation of proteins with ammonium sulfate; desalting on Sephadex
G-25; ion-exchange chromatography on DEAE-cellulose. The results showed an increased glutathione
reductase activity in blood serum of patients with impaired liver function. At the same time, the enzyme
activity in the blood serum of rats with toxic hepatitis varied similarly and correlated with this parameter
in the animals liver. To determine the possibility of modifying of the glutathione reductase conformation
under oxidative stress the enzyme purification from the experimental rats liver and study of its regulation by
intermediates of the pentose phosphate pathway - main source of NADPH for glutathione reductase reaction,
have been carried out. The detected differences of regulatory properties of glutathione reductase in health and
under toxic hepatitis suggest that one of the mechanisms of enzyme activity changes at liver pathologies of
different etiology may be conformational changes of its molecule.
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