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

Çiğdem Çoban; Yusuf Temel; Mehmet Çiftci

Article Details

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

Authors:

Çiğdem Çoban | Yusuf Temel | Mehmet Çiftci

Publisher:

Bingol University

Main Topics:

Enzimler

Journal:

Türk Doğa ve Fen Dergisi Vol.13 Issue 1, pp.62-69

Document Type:

Article

Publication Date:

3/2024

Document Language:

English

Document ID (Org No.):

2001314167

pISSN:

2149-6366

Keywords:

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 EUmg.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 enzymes affinity for GSSG and NADPH substrates. It was determined as KM constant 0.0061 mM and Vmax value 0.259 EUmL, for NADPH, KM constant was determined as 0.351 mM and Vmax value was determined as 0.604 EUmL 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 0.4660.387 mM competitive, 1.0570.110 mM non-competitive, 3.3861.305 mM competitive, 4.9100.960 mM competitive, and 20.7708.169 mM non-competitive, respectively.

 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 EUmg.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 enzymes affinity for GSSG and NADPH substrates. It was determined as KM constant 0.0061 mM and Vmax value 0.259 EUmL, for NADPH, KM constant was determined as 0.351 mM and Vmax value was determined as 0.604 EUmL 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 0.4660.387 mM competitive, 1.0570.110 mM non-competitive, 3.3861.305 mM competitive, 4.9100.960 mM competitive, and 20.7708.169 mM non-competitive, respectively.

 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 EUmg.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 enzymes affinity for GSSG and NADPH substrates. It was determined as KM constant 0.0061 mM and Vmax value 0.259 EUmL, for NADPH, KM constant was determined as 0.351 mM and Vmax value was determined as 0.604 EUmL 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 0.4660.387 mM competitive, 1.0570.110 mM non-competitive, 3.3861.305 mM competitive, 4.9100.960 mM competitive, and 20.7708.169 mM non-competitive, respectively.

 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 EUmg.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 enzymes affinity for GSSG and NADPH substrates. It was determined as KM constant 0.0061 mM and Vmax value 0.259 EUmL, for NADPH, KM constant was determined as 0.351 mM and Vmax value was determined as 0.604 EUmL 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 0.4660.387 mM competitive, 1.0570.110 mM non-competitive, 3.3861.305 mM competitive, 4.9100.960 mM competitive, and 20.7708.169 mM non-competitive, respectively.

Article Details

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

Authors:

Çiğdem Çoban | Yusuf Temel | Mehmet Çiftci

Publisher:

Bingol University

Main Topics:

Enzimler

Journal:

Türk Doğa ve Fen Dergisi Vol.13 Issue 1, pp.62-69

Document Type:

Article

Publication Date:

3/2024

Document Language:

English

Document ID (Org No.):

2001314167

pISSN:

2149-6366

Glutathione reductase

sheep

spleen

purification

antibiotic

inhibition

Issues

Article Details

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

Authors: Çiğdem Çoban | Yusuf Temel | Mehmet Çiftci

Publisher: Bingol University

Main Topics: Enzimler

Journal: Türk Doğa ve Fen Dergisi Vol.13 Issue 1, pp.62-69

Document Type: Article

Publication Date: 3/2024

Document Language: English

Document ID (Org No.): 2001314167

pISSN: 2149-6366

Glutathione reductase

sheep

spleen

purification

antibiotic

inhibition

Issues

Authors:

Çiğdem Çoban | Yusuf Temel | Mehmet Çiftci

Publisher:

Bingol University

Main Topics:

Enzimler

Journal:

Türk Doğa ve Fen Dergisi Vol.13 Issue 1, pp.62-69

Document Type:

Article

Publication Date:

3/2024

Document Language:

English

Document ID (Org No.):

2001314167

pISSN:

2149-6366