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Abstract

In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.1.44; 6PGD) was purified in human erythrocytes. This process was carried out by the preparation of hemolysate, precipitation by (NH4)2SO4 and 2',5'-ADP Sepharose 4B affinity chromatography . The degree of purity of the enzyme was determined with SDS-PAGE electrophoresis. The effect of ketoprofen on the enzyme was investigated in vitro and in vivo. Human erythrocyte 6PGD was purified in 742-fold at the end of all purification processes. The recovery of 6PGD was 50%, and its specific activity was 0.46U/mg in erythrocytes. Enzyme activity was spectrophotometrically measured using the Beutler method at 340 nm. Ketoprofen inhibited the enzyme activity in in vitro conditions. IC50 value of the drug inhibition in vitro was determined. For the drug having low IC50value (drug concentrations which produce 50% inhibition) (ketoprofen), in vivo studies were performed in New Zealand albino rabbits. In the evaluation of the in vivo effect of drug on 6PGD activity, it was observed that ketoprofen given at the first (P<0.01) and third hour (P<0.01), significantly inhibited the 6PGD activity

Keywords

• 6-phosphogluconate dehydrogenase, Drug, Erythrocyte, Inhibition

Ketoprofenin 6-Fosfoglukonat Dehidrogenaz Aktivitesi Üzerine In Vitro ve In Vivo Etkisinin Araştırılması

Abstract

Bu çalışmada 6-fosfoglukonat dehidrogenaz (E.C.1.1.1.44;6PGD) enzimi insan eritrositlerinden saflaştırıldı. Saflaştırma işlemi hemolizatın hazırlanması, amonyum sülfat çöktürmesi ve 2’,5’-ADP Sepharose 4B afinite kromatografisi yöntemi ile gerçekleştirildi. Enzimin saflık derecesi SDS-PAGE elektroforez yöntemi ile belirlendi. Enzim üzerine ketoprofenin in vitro ve in vivo etkisi araştırıldı. Tüm saflaştırma işlemleri sonunda insan eritrosit 6PGD enzimi 742 kat saflaştırıldı. Eritrosit 6PGD enzimi %50 verimle, spesifik aktivite 0.46 U/mg olarak elde edildi. Enzim aktivitesi spektrofotometrik olarak 340 nm’de Beutler metoduna göre ölçüldü. Çalışmada kullanılan ketoprofen ilacının in vitro şartlarda enzim aktivitesini inhibe ettiği gözlendi. In vitro inhibe eden bu ilaca ait ΙC50 değeri düşük olarak belirlendi. Daha sonra ketoprofenin in vivo inhibisyon etkisini belirlemek amacıyla Yeni Zelanda albino türü tavşanlar ile çalışmalar yapıldı. İlacın 6PGD enzim aktivitesi üzerine in vivo etkisi incelendiğinde I. saatte (P<0.01) ve III. saatte (P<0.01) ketoprofen ilacının enzim aktivitesini istatistiksel olarak önemli derecede inhibe ettiği gözlendi. 

Keywords

• 6 fosfoglukonat dehidrogenaz, Eritrosit, İlaç, İnhibisyon

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