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

Yıl 2011, Cilt: 39 Sayı: 4, 413 - 419, 01.12.2011

Veysel Çomaklı Mehmet Çiftci Ömer İrfan Küfrevioğlu

Öz

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.

Anahtar Kelimeler

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

Öz

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

Anahtar Kelimeler

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

Kaynakça

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