Activity of glutathione reductase in type 2 diabetes mellitus and catalytic properties of the enzyme isolated using chromatographic methods
Abstract
The aim of this work was to determine the activity of glutathione reductase (GR, EC 1.6.4.2) in the blood serum of patients with type 2 diabetes mellitus (T2DM) and in the blood serum of rats with experimental T2DM, as well as to develop a scheme for purifying the enzyme from the liver of experimental animals using chromatographic methods and to study its catalytic properties. The experiment used the blood serum of practically healthy individuals with normal general and biochemical blood test results (control group of patients), people diagnosed with T2DM, as well as the serum and liver of rats in the control group and animals with experimental T2DM. The pathological condition in experimental animals was modeled by a combination of a high-fat diet for 1 month and subsequent double intraperitoneal administration of streptozotocin (STZ) at an interval of 7 days at a dose of 30 mg / kg of animal weight in citrate buffer pH 4.4. The animals were slaughtered 14 days after the administration of STZ. GR activity was determined spectrophotometrically on SF-56 at 340 nm. The total amount of protein was determined by the Lowry method. To study the catalytic properties of the enzyme, it was purified from the liver of rats in the control group and animals with induced T2DM using the methods of protein separation with ammonium sulfate, as well as gel filtration through Sephadex G-25 and ion-exchange chromatography on DEAE-cellulose. As a result, GR enzyme preparations were obtained, purified 56.4 and 46.0 times from the liver of rats in the control group and animals with T2DM. The study found that during ion-exchange chromatography on a column with DEAE-cellulose, GR from the liver of rats in the control group was desorbed with maximum enzymatic activity at a KCl concentration of 100 mM. For desorption of GR from the liver of rats with type 2 diabetes from the column, the most effective concentration of KCl in the elution medium was 50 mM. Using the obtained enzyme preparations, it was found that the kinetics of the reaction catalyzed by GR is described by the Michaelis-Menten equation. In this case, in pathology, there is a decrease in the affinity of the enzyme to both oxidized glutathione and NADPH. As a result of assessing the effect of pH on the intensity of GR functioning, it was found that this enzyme from rat hepatocytes normally has the highest activity at pH values from 7.2 to 7.5. The optimal pH value corresponded to 7.4. An increase or decrease in the concentration of hydrogen ions was accompanied by a sharp drop in enzymatic activity. It was shown that for the enzyme isolated from the liver of rats with diabetes, the pH optimum is 7.0. Moreover, a change in the concentration of hydrogen ions to a greater extent is accompanied by a less sharp drop in GR activity, compared to the norm.
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