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

Year 2024, , 62 - 69, 26.03.2024
https://doi.org/10.46810/tdfd.1333609

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.

Supporting Institution

Bingöl Üniversitesi Araştırma Projeleri Koordinasyon Birimi

Project Number

BAP-FEF-2019.00.007.

Thanks

We would like to thank Bingöl University Scientific Research Projects Coordination Unit (BÜBAP) for their financial support with the project numbered BAP-FEF-2019.00.007.

References

  • Winterbourn CC. Regulation of intracellular glutathione. Redox Biol 2019; 22: 101086.
  • Balendiran GK, Dabur R, Fraser D. The role of glutathione in cancer. Cell Biochem Funct 2004; 22(6): 343–52.
  • Temel Y, Kufrevioglu OI, Ciftci M. Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity. Turkish J Chem 2017;41(1): 48–60.
  • Taser P, Ciftci M. Purifi cation and characterization of glutathione reductase from turkey liver 2012; 36(5): 546–53.
  • Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem 2009; 390(3): 191–214.
  • Temel Y, Taysi MŞ. The Effect of Mercury Chloride and Boric Acid on Rat Erythrocyte Enzymes. Biol Trace Elem Res 2019; 191(1): 172–187.
  • Couto N, Wood J, Barber J. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radic Biol Med 2016; 95: 27–42.
  • Meldrum NU, Tarr HL. The reduction of glutathione by the Warburg-Christian system. Biochem. J. 1935; 29: 108-15
  • Can B, Kulaksiz Erkmen G, Dalmizrak O, Ogus I H, Ozer N. Purification and characterisation of rat kidney glutathione reductase. Protein J. 2010; 29: 250-256.
  • Kuzu M, Aslan A, Ahmed I, Comakli V, Demirdag R, Uzun N. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities. Fish Physiol Biochem 2016; 42(2): 483–91.
  • Ulusu NN, Tandoğan B. Purification and kinetic properties of glutathione reductase from bovine liver. Mol Cell Biochem 2007; 303(1–2): 45–51.
  • Hutchings M, Truman A, Wilkinson B. Antibiotics: past, present and future. Curr Opin Microbiol 2019;51:72–80.
  • Beydemir S, Ciftci M, Ozmen İ, Buyukkuroglu ME, Ozdemir H, Kufrevioglu OI. Effects of some medical drugs on enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Pharmacol.Res 2000; 42: 187-191.
  • Erat M, Sakiroglu H, Ciftci M. Effects of some Antibiotics on Glutathione Reductase from Bovine Erythrocytes. Vet. Med. Czech 2003; 48(11): 305–312.
  • Temel Y, Bozkus T, Karagözoglu Y, Ciftci M. Purification and Characterization of Glutathion Reductase Enzyme From Japanese Quail ( Coturnix coturnix japanica ) 2017; 7(3): 143–50.
  • Carlberg I, Mannervik B. Purification and characterization of glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2’, 5’-ADP Sepharose-4B. Anal. Biochem 1981; 116:531–536.
  • Smith LL. Cholesterol autoxidation. Chem. Phys. Lipids 1987; 44: 87-125.
  • Adem S, Ciftci M. Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2’,5’-ADP Sepharose-4B affinity in a single chromatography step. Protein Expr Purif 2012; 81(1):1-4.
  • Temel Y, Kocyigit UM. Purification of glucose-6-phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity. J Biochem Mol Toxicol 2017; 31(9): e21927.
  • Laemmli DK. Cleavage of structural proteins during in assembly of the heat ofbacteriophage T4. Nature, London, 1970 s. 227-680.
  • Lineweaver H, Burk D The determination of enzyme dissociation constants. J Amer Chem Soc 1934; 56(3):658–666.
  • Keha E, Kufrevioglu OI. Biyokimya, Aktif Yayınevi, Erzurum; 2010, s. 653.
  • Tekman B, Ozdemir H, Senturk M, Ciftci M. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity. Comparative Biochemistry and Physiology 2008; 148: 117-121.
  • Erat M, Ciftci M. Effect of Melatonin on Enzyme Activities of Glutathione Reductase from Human Erythrocytes In Vitro and from Rat Erythrocytes In Vivo. Eur.J. Phlogy 2006; 537: 59-63.
  • Güller P. Gentamisin, amoksisilin ve sefazolin sodyum antibiyotiklerinin fare karaciğer ve böbrek glutatyon redüktaz enziminin aktivite, protein ve gen ekspresyon düzeyleri üzerine etkilerinin incelenmesi. Kimya anabilim dalı, Atatürk üniversitesi, Erzurum, Türkiye, 2015.
  • Ulusu G, Erat M, Ciftci M, Sakiroglu H, Bakan E. Purification and characterization of glutathione reductase from sheep liver. Turkish Journal of Veterinary and Animal Sciences 2005; 29(5): 1109-1117.
  • Erat M, Ciftci M. In vitro effects of some antibiotics on glutathione reductase from sheep liver. Journal of Enzyme and Medicinal Chemistry 2003; 18: 545-550.
  • Erat M. Purification of human and bovine erythrocyte glutathione reductase enzyme, investigation of the inhibition or activation effects of some drugs and chemicals. Department of Chemistry, Atatürk University, Erzurum, Turkiye, 2002.
  • Bilir G. Purification of glutathione reductase enzyme from soybean seed (glycine max l.) and examination of the inhibition kinetics of some heavy metals. Department of agricultural biotechnology, Ondokuz Mayıs University, Samsun, Turkiye, 2017.
  • Kavutçu M. Isolation, purification and investigation of physicochemical and kinetic properties of adenosine deamylase enzyme from sheep spleen. Department of Biochemistry, Ankara University, Ankara, Türkiye, 1995.
  • Temel Y, Ayna A, Hamdi Shafeeq I, Ciftci M. In vitro effects of some antibiotics on glucose-6-phosphate dehydrogenase from rat (Rattus norvegicus) erythrocyte. Drug and chemical toxicology 2020; 43(2): 219-223.
  • Bayindir S, Ayna A, Temel Y, Ciftci M. The synthesis of new oxindoles as analogs of natural product 3, 3′-bis (indolyl) oxindole and in vitro evaluation of the enzyme activity of G6PD and 6PGD. Turkish Journal of Chemistry 2018; 42(2):332-345.
  • Ayna A, Khosnaw L, Temel Y, Ciftci M. Antibiotics as inhibitor of glutathione S-transferase: biological evaluation and molecular structure studies. Current drug metabolism 2021; 22(4): 308-314.
Year 2024, , 62 - 69, 26.03.2024
https://doi.org/10.46810/tdfd.1333609

Abstract

Project Number

BAP-FEF-2019.00.007.

References

  • Winterbourn CC. Regulation of intracellular glutathione. Redox Biol 2019; 22: 101086.
  • Balendiran GK, Dabur R, Fraser D. The role of glutathione in cancer. Cell Biochem Funct 2004; 22(6): 343–52.
  • Temel Y, Kufrevioglu OI, Ciftci M. Investigation of the effects of purification and characterization of turkey (Meleagris gallopavo) liver mitochondrial thioredoxin reductase enzyme and some metal ions on enzyme activity. Turkish J Chem 2017;41(1): 48–60.
  • Taser P, Ciftci M. Purifi cation and characterization of glutathione reductase from turkey liver 2012; 36(5): 546–53.
  • Ballatori N, Krance SM, Notenboom S, Shi S, Tieu K, Hammond CL. Glutathione dysregulation and the etiology and progression of human diseases. Biol Chem 2009; 390(3): 191–214.
  • Temel Y, Taysi MŞ. The Effect of Mercury Chloride and Boric Acid on Rat Erythrocyte Enzymes. Biol Trace Elem Res 2019; 191(1): 172–187.
  • Couto N, Wood J, Barber J. The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radic Biol Med 2016; 95: 27–42.
  • Meldrum NU, Tarr HL. The reduction of glutathione by the Warburg-Christian system. Biochem. J. 1935; 29: 108-15
  • Can B, Kulaksiz Erkmen G, Dalmizrak O, Ogus I H, Ozer N. Purification and characterisation of rat kidney glutathione reductase. Protein J. 2010; 29: 250-256.
  • Kuzu M, Aslan A, Ahmed I, Comakli V, Demirdag R, Uzun N. Purification of glucose-6-phosphate dehydrogenase and glutathione reductase enzymes from the gill tissue of Lake Van fish and analyzing the effects of some chalcone derivatives on enzyme activities. Fish Physiol Biochem 2016; 42(2): 483–91.
  • Ulusu NN, Tandoğan B. Purification and kinetic properties of glutathione reductase from bovine liver. Mol Cell Biochem 2007; 303(1–2): 45–51.
  • Hutchings M, Truman A, Wilkinson B. Antibiotics: past, present and future. Curr Opin Microbiol 2019;51:72–80.
  • Beydemir S, Ciftci M, Ozmen İ, Buyukkuroglu ME, Ozdemir H, Kufrevioglu OI. Effects of some medical drugs on enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Pharmacol.Res 2000; 42: 187-191.
  • Erat M, Sakiroglu H, Ciftci M. Effects of some Antibiotics on Glutathione Reductase from Bovine Erythrocytes. Vet. Med. Czech 2003; 48(11): 305–312.
  • Temel Y, Bozkus T, Karagözoglu Y, Ciftci M. Purification and Characterization of Glutathion Reductase Enzyme From Japanese Quail ( Coturnix coturnix japanica ) 2017; 7(3): 143–50.
  • Carlberg I, Mannervik B. Purification and characterization of glutathione reductase from calf liver. An improved procedure for affinity chromatography on 2’, 5’-ADP Sepharose-4B. Anal. Biochem 1981; 116:531–536.
  • Smith LL. Cholesterol autoxidation. Chem. Phys. Lipids 1987; 44: 87-125.
  • Adem S, Ciftci M. Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2’,5’-ADP Sepharose-4B affinity in a single chromatography step. Protein Expr Purif 2012; 81(1):1-4.
  • Temel Y, Kocyigit UM. Purification of glucose-6-phosphate dehydrogenase from rat (Rattus norvegicus) erythrocytes and inhibition effects of some metal ions on enzyme activity. J Biochem Mol Toxicol 2017; 31(9): e21927.
  • Laemmli DK. Cleavage of structural proteins during in assembly of the heat ofbacteriophage T4. Nature, London, 1970 s. 227-680.
  • Lineweaver H, Burk D The determination of enzyme dissociation constants. J Amer Chem Soc 1934; 56(3):658–666.
  • Keha E, Kufrevioglu OI. Biyokimya, Aktif Yayınevi, Erzurum; 2010, s. 653.
  • Tekman B, Ozdemir H, Senturk M, Ciftci M. Purification and characterization of glutathione reductase from rainbow trout (Oncorhynchus mykiss) liver and inhibition effects of metal ions on enzyme activity. Comparative Biochemistry and Physiology 2008; 148: 117-121.
  • Erat M, Ciftci M. Effect of Melatonin on Enzyme Activities of Glutathione Reductase from Human Erythrocytes In Vitro and from Rat Erythrocytes In Vivo. Eur.J. Phlogy 2006; 537: 59-63.
  • Güller P. Gentamisin, amoksisilin ve sefazolin sodyum antibiyotiklerinin fare karaciğer ve böbrek glutatyon redüktaz enziminin aktivite, protein ve gen ekspresyon düzeyleri üzerine etkilerinin incelenmesi. Kimya anabilim dalı, Atatürk üniversitesi, Erzurum, Türkiye, 2015.
  • Ulusu G, Erat M, Ciftci M, Sakiroglu H, Bakan E. Purification and characterization of glutathione reductase from sheep liver. Turkish Journal of Veterinary and Animal Sciences 2005; 29(5): 1109-1117.
  • Erat M, Ciftci M. In vitro effects of some antibiotics on glutathione reductase from sheep liver. Journal of Enzyme and Medicinal Chemistry 2003; 18: 545-550.
  • Erat M. Purification of human and bovine erythrocyte glutathione reductase enzyme, investigation of the inhibition or activation effects of some drugs and chemicals. Department of Chemistry, Atatürk University, Erzurum, Turkiye, 2002.
  • Bilir G. Purification of glutathione reductase enzyme from soybean seed (glycine max l.) and examination of the inhibition kinetics of some heavy metals. Department of agricultural biotechnology, Ondokuz Mayıs University, Samsun, Turkiye, 2017.
  • Kavutçu M. Isolation, purification and investigation of physicochemical and kinetic properties of adenosine deamylase enzyme from sheep spleen. Department of Biochemistry, Ankara University, Ankara, Türkiye, 1995.
  • Temel Y, Ayna A, Hamdi Shafeeq I, Ciftci M. In vitro effects of some antibiotics on glucose-6-phosphate dehydrogenase from rat (Rattus norvegicus) erythrocyte. Drug and chemical toxicology 2020; 43(2): 219-223.
  • Bayindir S, Ayna A, Temel Y, Ciftci M. The synthesis of new oxindoles as analogs of natural product 3, 3′-bis (indolyl) oxindole and in vitro evaluation of the enzyme activity of G6PD and 6PGD. Turkish Journal of Chemistry 2018; 42(2):332-345.
  • Ayna A, Khosnaw L, Temel Y, Ciftci M. Antibiotics as inhibitor of glutathione S-transferase: biological evaluation and molecular structure studies. Current drug metabolism 2021; 22(4): 308-314.
There are 33 citations in total.

Details

Primary Language English
Subjects Enzymes
Journal Section Articles
Authors

Çiğdem Çoban 0000-0003-1141-544X

Yusuf Temel 0000-0001-8148-3718

Mehmet Çiftci 0000-0002-1748-3729

Project Number BAP-FEF-2019.00.007.
Early Pub Date March 26, 2024
Publication Date March 26, 2024
Published in Issue Year 2024

Cite

APA Çoban, Ç., Temel, Y., & Çiftci, M. (2024). Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity. Türk Doğa Ve Fen Dergisi, 13(1), 62-69. https://doi.org/10.46810/tdfd.1333609
AMA Çoban Ç, Temel Y, Çiftci M. Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity. TDFD. March 2024;13(1):62-69. doi:10.46810/tdfd.1333609
Chicago Çoban, Çiğdem, Yusuf Temel, and Mehmet Çiftci. “Purification, Characterization of Glutathion Reductase Enzyme from Sheep Splen Tissue and investıgatıon of the Effects of Some Antibiotics on Enzyme Activity”. Türk Doğa Ve Fen Dergisi 13, no. 1 (March 2024): 62-69. https://doi.org/10.46810/tdfd.1333609.
EndNote Çoban Ç, Temel Y, Çiftci M (March 1, 2024) Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity. Türk Doğa ve Fen Dergisi 13 1 62–69.
IEEE Ç. Çoban, Y. Temel, and M. Çiftci, “Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity”, TDFD, vol. 13, no. 1, pp. 62–69, 2024, doi: 10.46810/tdfd.1333609.
ISNAD Çoban, Çiğdem et al. “Purification, Characterization of Glutathion Reductase Enzyme from Sheep Splen Tissue and investıgatıon of the Effects of Some Antibiotics on Enzyme Activity”. Türk Doğa ve Fen Dergisi 13/1 (March 2024), 62-69. https://doi.org/10.46810/tdfd.1333609.
JAMA Çoban Ç, Temel Y, Çiftci M. Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity. TDFD. 2024;13:62–69.
MLA Çoban, Çiğdem et al. “Purification, Characterization of Glutathion Reductase Enzyme from Sheep Splen Tissue and investıgatıon of the Effects of Some Antibiotics on Enzyme Activity”. Türk Doğa Ve Fen Dergisi, vol. 13, no. 1, 2024, pp. 62-69, doi:10.46810/tdfd.1333609.
Vancouver Çoban Ç, Temel Y, Çiftci M. Purification, characterization of glutathion reductase enzyme from sheep splen tissue and investıgatıon of the effects of some antibiotics on enzyme activity. TDFD. 2024;13(1):62-9.