Purification of Glutathione S-Transferase Enzyme from Rainbow Trout Erythrocytes and Examination of the Effects of Certain Antibiotics on Enzyme Activity

Year 2011, Volume: 39 Issue: 4, 413 - 419, 01.12.2011

Veysel Çomaklı Mehmet Çiftci Ömer İrfan Küfrevioğlu

Abstract

The present study investigated the effects of certain antibiotics on the enzyme activity of glutathione- S-transferase EC 2.5.1.18 obtained from rainbow trout erythrocytes. For this purpose, erythrocyte glutathione S-transferase enzyme was purified 8714-fold by glutathione–agarose affinity chromatography with a yield of 90%. Temperature was kept under control 4ºC during purification. Enzyme purification was checked by performing SDS-PAGE. A single band was obtained approximately at 23 kDa. GST enzyme activity was determined by spectroscopic monitoring of the formation of 1-chloro-2,4-dinitrobenzene-glutathione CDNB- GSH conjugate. The same method was used for all kinetic studies. Furthermore, by examining the inhibitory effects upon enzyme activity of antibiotics such as gentamicin, amikacin, cefuroxime sodium, ampicillin, ornidazole, and metranidazole, the IC50 values and Ki constants were calculated and inhibition types were identified for gentamicin, amikacin, and cefuroxime sodium, medications that displayed high inhibitory effects.

Keywords

Glutathione-S-transferase EC 2.5.1.18, rainbow trout erythrocytes, antibiotics

Gökkuşağı Alabalık Eritrositlerinden Glutatyon S-Transferas Enziminin Saflaştırılması ve Bazı Antibiyotiklerin Enzim Aktivitesi Üzerine Etkilerinin İncelenmesi

Abstract

B u çalışmada bazı antibiyotiklerin gökkuşağı alabalık eritrositlerinden elde edilen glutatyon-S-transferaz EC 2.5.1.18 enzim aktivitesi üzerine etkileri araştırıldı. Bu amaçla eritrosit glutatyon S- transferaz enzimi glutatyon–agaroz afinite kromatografisi tekniği ile %90 verimle 8714 kat saflaştırıldı. Saflaştırma esnasında sıcaklık kontrol altında tutuldu +4oC . Enzim saflığı SDS-PAGE yapılarak kontrol edildi. Yaklaşık olarak 23 kDa’da tek bant elde edildi. GST enzim aktivitesi, 1-kloro-2,4-dinitrobenzenglutatyon CDNB-GSH konjugatının oluşumunun spektroskopik olarak izlenmesi ile belirlendi. Kinetik çalışmaların tamamında bu metot kullanıldı. Ayrıca enzim aktivitesi üzerine gentamisin, amikasin, sefuroksim sodyum, ampisilin, ornidazol ve metranidazol gibi antibiyotiklerin inhibisyon etkileri incelenerek yüksek inhibisyon etkisi gösteren gentamisin, amikasin, sefuroksim sodyum ilaçlarının IC değerleri ve Ki sabitleri hesaplanarak inhibisyon tipleri belirlendi

Keywords

Glutatyon-S-transferaz EC 2.5.1.18, gökkuşağı alabalık eritrositleri, antibiyotikler

References

• 1. S. Güvercin, M. Erat, H. Şakiroğlu, Determination of Some Kinetic and Characteristic Properties of Glutathione S-transferase from Bovine Erythrocytes, Protein & Peptide Letters, 15 (1) (2008) 6.
• 2. J.D. Hayes, J.U. Flanagan, I.R. Jowsey, Glutathione Transferases. Annu. Rev. Pharmacol. Toxicol. 45 (2005) 51.
• 3. R.C. Strange, M.A. Spiteri, S. Ramachandran, A.A. Fryer, Glutathione-S-transferase family of enzymes. Mutation Research., 482 (2001) 21.
• 4. R.N. Armstrong, Structure, catalytic mechanism, and evolution of the glutathione transferases, Chem. Res. Toxicol., 10(1) (1997) 2.
• 5. F.S. Kılıç, D. Kaya, N. Kiraz, İ. Cingi, Aminoglikozid antibiyotiklerin antibakteriyel aktivitelerine S-Karboksimetil-sisteinin etkisi. Ankem derg 9 No.1 (1995) 76.
• 6. S.O. Kayaalp, Rasyonel tedavi yönünden tıbbi farmakoloji.10. Baskı. (2000)
• 7. A.A. Hunaiti, and M. Soud, Effect of lead concentrationon the level of glutathione, glutathione S-transferase, reductase and peroksidase in human blood. The Science of The Total Environment, 248 (2000) 45.
• 8. F. Toribio, E. Martinez-Lara, P. Pascual, J. Lopez-Barea, Methods for purification of glutathione peroksidase and related enzymes, Journal of Chromatography B., 684 (1996) 77.
• 9. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72 (1976) 248.
• 10. D.K. Laemmli, Cleavage of structural proteins during in assembly of the head of Bacteriophage T4. Nature. 227 (1970) 680.
• 11. H. Lineweaver, D. Burk, The determination of enzyme dissociation constants. J. Am. Chem. Soc., 57 (1934) 685.
• 12. M. Şentürk, Ö.İ. Küfrvioğlu, M. Çiftçi, Effects of some antibiotics on human erythrocyte glutathione reductase: an in vitro study. J Enzyme Inhib Med Chem. Feb;23(1) (2008)144.
• 13. G.J. Smith, V.S. Ohl, G. Litwack, The glutathione stransferases and chemically induced hepatocarcinogenesis. Cancer Res., 37 (1977) 8.
• 14. E. Hodgson, P.E. Levi, Introduction to Biochemical Toxicology. Connecticut: Appleton&Lange, 1994.
• 15. R. Morgenstren, G. Lundqvist, V. Hancock, J.W. De Pierre, Studies on the activity and activation of rat liver microsomal glutathione transferase, in particular with a substrate analogue series. J Biol Chem, 263 (1988) 6671.
• 16. B. Ketterer, Detoxication reactions of glutathione and glutathione transferases. Xenobiotica. 16 (1986) 957.
• 17. N.P.E. Vermeulen, Analysis of mercapturic acids as a tool in biotransformation, biomonitoring and toxicological studies. “Glutathione-S-transferases and Drug Resistance” Taylor and Francis London. 1 (1990) 141.
• 18. V.K. Çelik, F. Armutcu, A. Aker, Human Placental Glutath-ione-S-Transferase: Isolation, Kinetic Properties and in vitro Interactions with Several Drugs. C. Ü. Tıp Fakültesi Dergisi. 25(4) (2003)187.
• 19. T. Bucciarelli, P. Sacchetta, A. Pennelli, L. Cornelio, R. Romagnoli, S. Melino, R. Petruzzelli, C. Di İlio, Characterization Of Toad Liver Glutathine Transferase. Biochimica Et Biophysica Acta. 1431 (1999) 189.
• 20. B. Mannervik, U. H. Danielson, Glutathione transferases- structure and catalytic activity. Dep. of Biochemistry. 23(3) (1988) 283.
• 21. M. Çiftçi, V. Türkoğlu, T.A. Çoban, Effects of some drugs on hepatic glucose 6-phosphate dehydrogenase activity in Lake Van Fish (Chalcalburnus Tarischii Pallas, 1811). Journal of Hazardous Materials, 143 (2007) 415.
• 22. M. Erat, H. Şakiroğlu, M. Çiftçi, Effects of some antibiotics on glutathione reductase activities from human erythrocytes in vitro and from rat erythrocytes in vivo. J Enzyme Inhib Med Chem. 20(1) (2005) 69.
• 23. M. Erat, M. Çiftçi, In Vitro Effects of Some Antibiotics on Glutathione Reductase from Sheep Liver. Journal of Enzyme Inhibition and Medicinal Chemistry, 18 (6) (2003) 545.
• 24. O. Erdoğan, M. Çiftci, A. Çiltaş, O. Hisar, Inhibition Effects of Some Antibiotics on the Activity of Glucose 6-Phosphate Dehydrogenase Enzyme from Rainbow Trout (Oncorhynchus mykiss Walbaum, 1792) Erythrocytes Turk J Vet Anim Sci. 28 (2004) 675.
• 25. M. Akyüz, M. Erat, M. Çiftçi, K. Gümüştekin, N. Bakan, Effects of some antibiotics on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro and in vivo study. J Enzyme Inhib Med Chem. 54 (2004) 275.