Veysel ÇOMAKLI, Ö. İrfan KÜFREVİOĞLU, Mehmet ÇİFTÇİ

Purification of glutathione S-transferase enzyme from rainbow trout erythrocytes and examination of the effects of certain antibiotics on enzyme activity

Bu 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 $(+4^{o}C).$). 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 $IN_{50}$ değerleri ve Ki sabitleri hesaplanarak inhibisyon tipleri belirlendi.

Gökkuşağı alabalık eritrositlerinden glutatyon S-transferaz enziminin saflaştırılması ve bazı antibiyotiklerin enzim aktivitesi üzerine etkilerinin incelenmesi

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 $(+4^{o}C).$ 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 $IN_{50}$ values and Ki constants were calculated and inhibition types were identified for gentamicin, amikacin, and cefuroxime sodium, medications that displayed high inhibitory effects.

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