The activity of glucose-6-phosphate dehydrogenase in rat liver under oxidative stress development and application of chromatography different types for enzyme purification and its properties study
Abstract
The aim of the work was to estimate the activity of glucose-6-phosphate dehydrogenase (G6PD) in
the liver of rats with experimental toxic hepatitis (ETG), rheumatoid arthritis (RA) and hyperthyroidism (HT)
and to investigate some properties of the enzyme using purified preparations from rats liver tissue at the
control and ETG as model systems. G6PD preparations have been obtained by chromatographic techniques
combination. The induction of ETG has been implemented by CCl4 use, PA – by complete Freund's adjuvant,
GT – by triiodothyronine. G6PD activity was determined by spectrophotometry at 340 nm. Isolation and
purification of the enzyme in control and ETG have been carried out by mechanical homogenization of the
liver tissue, ammonium sulfate fractionation, gel-filtration over Sephadex G-25, ion-exchange
chromatography on DEAE-cellulose and gel-chromatography through Sephadex G-150. The increased G6PD activity has been found in the liver of animals under the development of pathologies accompanied by
oxidative stress, that can be of great adaptive significance in relation to the need of NADPH supply for the
glutathione antioxidant system functioning. Thus, it has been shown that G6PD activity expressed as U/g of
wet weight increased in the liver of rats with ETG 1.5 times, under RA - 1.2 times, at HT - 1.3 times. The
highly purified enzyme preparations have been obtained from the liver of control rats and animals with ETG
with purification degree of 108.8 and 100.4, specific activity of 4.133 and 6.529 U/protein mg and yield of
8.2% and 8.3% respectively. With their use it has been shown that ATP, ADP and AMP affected in varying
degrees on the analyzed enzyme in normal and pathological conditions, apparently due to the modification of
its structure under oxidative stress. We can not exclude that some changes may play a role in the limitation of
the reactive oxygen species level by NADPH supply for glutathione antioxidant system.
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