Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity

Abstract

In this study, glutathione reductase (EC 1.8.1.7; GR, Glutathione: NADP+ oxidoreductase), which is the key enzyme of antioxidant metabolism, was purified from sheep spleen using ammonium sulfate precipitation and 2', 5'-ADP Sepharose-4B affinity chromatography. As a result of purification, the GR enzyme was purified, with 20.03 EU/mg.protein of specific activity, 1564.8 times a yield of 40.61%. The purity of the enzyme was checked by SDS-PAGE. In the characterization studies, optimum pH, optimum ionic strength, stable pH, optimum temperature and subunit molecular mass of the enzyme were determined. In addition, KM and Vmax values were found to determine the enzyme's affinity for GSSG and NADPH substrates. It was determined as KM constant 0.0061 mM and Vmax value 0.259 EU/mL, for NADPH, KM constant was determined as 0.351 mM and Vmax value was determined as 0.604 EU/mL for GSSG. In addition, the effects of ampicillin, streptomycin sulfate, gentamicin, cefoperazone sodium and precort-lyo on enzyme activity were investigated. It was determined that these drugs showed an inhibitory effect on GR enzyme activity purified from sheep spleen tissue. The IC50 values for precort-lyo, ampicillin, streptomycin sulfate, cefoperazone sodium, and gentamicin were 1.27 mM, 3.22 mM, 7.95 mM, 16.97 mM, and 17.20 Mm, respectively; and Ki constants were calculated as 0.466±0.387 mM (competitive), 1.057±0.110 mM (non-competitive), 3.386±1.305 mM (competitive), 4.910±0.960 mM (competitive), and 20.770±8.169 mM (non-competitive), respectively.

Keywords

• Glutathione reductase
• sheep
• spleen
• purification
• antibiotic
• inhibition

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