Öz
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
Anahtar Kelimeler
6-phosphogluconate dehydrogenase Drug Erythrocyte Inhibition
Kaynakça
- Akyüz M., Erat M., Çiftçi M., Gümüştekin K., Bakan N., 2004. Effects of some antibiotics on human erythrocyte 6-Phospho Gluconate cleavage of liver 6-P-gluconate presence of vanadate. Archives of Biochemistry and Biophysics, 321, 1-5.
- Beutler E., 1971. Redcell metabolism. Manual of biochemical methods. 12, 68-70, Academic press, London.
- Beydemir Ş., Çiftçi M., Özmen İ., Büyükokuroğlu ME., Özdemir H., Küfrevioğlu Öİ., 2000. Effects of some medical drugs on enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Pharmacological Research, 42, 187-191.
- Beydemir Ş., Gülçin I., Kufrevioglu OI., Ciftci M., 2003. Glucose 6-phosphate dehydrogenase: In vitro and in vivo effects of dantrolene sodium. Polish Journal of Pharmacology, 55, 787-792.
- Beydemir Ş., Çiftçi M., Yılmaz H., Küfrevioğlu OI., 2004. 6-Phosphogluconate Dehydrogenase: Purification, Characterization and Kinetic Properties from Rat Erythrocytes. Turkish Journal of Veterinary&Animal Sciences, 28, 707-714.
- Bradford MM., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 7, 248-254.
- Caprari P., Caforio MP., Cianciulli P., Maffi D., Pasquino MT., Tarzia A., Amadori S., Salvati AM., 2001. 6-Phosphogluconate
- dehydrogenase deficiency in an Italian family.
- Annals of Hematology, 80, 41-44.
- Keha E., Küfrevioğlu Öİ., 2004. Biyokimya, Aktif Yayınevi, Erzurum.
- Laemmli DK., 1970. Cleavage of structural proteins during in assembly of the head of bacteriophage T4. Nature, 227, 680-685.
- Lehninger AL., 1993. Principles of Biochemistry. 2nd ed., Worth Publishers, New York.
- Mulcahy DM., Tuomi P., Larsen RS., 2003. Differential mortality of male spectacled eiders (Somateria fischeri) and king eiders (Somateria spectabilis) subsequent to anesthesia with propofol, bupivacaine, and ketoprofen. Journal of Avian Medicine and Surgery, 17, 117-123.
- Ninfali P., Orsenigo T., Barociani SR., 1990. Rapid purification of glucose 6-phosphogluconate dehydrogenase from mammal’s erythrocytes. Preparative Biochemistry and Biotechnology, 20, 297-309.
- Özabacıgil F., 2005. İnsan eritrosit 6-fosfoglukonat dehidrogenaz enziminin saflaştırılması, bazı ilaçların enzim aktivitesi üzerine in vitro ve tavşanlarda in vivo etkisinin incelenmesi. Atatürk Üniversitesi, Sağlık Bilimleri Enstitüsü, Erzurum, Türkiye.
- Ozabacigil F., Beydemir Ş., Çiftçi M., Gümüştekin K., Bakan N., 2008. Cisplatin and 5-fluorouracil inhibits 6-phosphogluconate dehydrogenase activity in human erythrocytes in vitro and in vivo. Asian Journal of Chemistry, 20, 3189- 3196.
- Puskas F., Gergely P., Banki K., Perl A., 2000. Stimulation of the pentose phosphate pathway and glutathione levels by dehydroascorbate, the oxidized form of vitamin C. The Journal of the Fedaration of American Societies for Experimental Biology, 14, 1352-1361.
- Segel IH., 1976. Biochemical calculations. John Wiley and Sons, Inc., 403, New York.
- Shreve DS., Levy HR., 1977. On the molecular weight of human glucose 6-phosphate dehydrogenase. Biochemical and Biophysical Research Communications, 78, 1369-1375.
- Tandoğan B., Ulusu N., 2003. Review. 6- Fosfoglukonat dehidrogenaz: Moleküler ve kinetik özellikler. Türk Biyokimya Dergisi, 28, 268-273.
- Walzem RL., Storebakken T., Hung SSO., Hansen RJ., 1991. Relationship between growth and selected liver enzyme activities of individual rainbow trout. Journal of Nutrition, 121, 1090- 1098.
- Williams RL., Upton RA., 1998. The clinical pharmacology of ketoprofen. Journal of Clinical Pharmacology, 28, 13-22.
Ketoprofenin 6-Fosfoglukonat Dehidrogenaz Aktivitesi Üzerine In Vitro ve In Vivo Etkisinin Araştırılması
Öz
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.
Anahtar Kelimeler
Kaynakça
- Akyüz M., Erat M., Çiftçi M., Gümüştekin K., Bakan N., 2004. Effects of some antibiotics on human erythrocyte 6-Phospho Gluconate cleavage of liver 6-P-gluconate presence of vanadate. Archives of Biochemistry and Biophysics, 321, 1-5.
- Beutler E., 1971. Redcell metabolism. Manual of biochemical methods. 12, 68-70, Academic press, London.
- Beydemir Ş., Çiftçi M., Özmen İ., Büyükokuroğlu ME., Özdemir H., Küfrevioğlu Öİ., 2000. Effects of some medical drugs on enzyme activities of carbonic anhydrase from human erythrocytes in vitro and from rat erythrocytes in vivo. Pharmacological Research, 42, 187-191.
- Beydemir Ş., Gülçin I., Kufrevioglu OI., Ciftci M., 2003. Glucose 6-phosphate dehydrogenase: In vitro and in vivo effects of dantrolene sodium. Polish Journal of Pharmacology, 55, 787-792.
- Beydemir Ş., Çiftçi M., Yılmaz H., Küfrevioğlu OI., 2004. 6-Phosphogluconate Dehydrogenase: Purification, Characterization and Kinetic Properties from Rat Erythrocytes. Turkish Journal of Veterinary&Animal Sciences, 28, 707-714.
- Bradford MM., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry, 7, 248-254.
- Caprari P., Caforio MP., Cianciulli P., Maffi D., Pasquino MT., Tarzia A., Amadori S., Salvati AM., 2001. 6-Phosphogluconate
- dehydrogenase deficiency in an Italian family.
- Annals of Hematology, 80, 41-44.
- Keha E., Küfrevioğlu Öİ., 2004. Biyokimya, Aktif Yayınevi, Erzurum.
- Laemmli DK., 1970. Cleavage of structural proteins during in assembly of the head of bacteriophage T4. Nature, 227, 680-685.
- Lehninger AL., 1993. Principles of Biochemistry. 2nd ed., Worth Publishers, New York.
- Mulcahy DM., Tuomi P., Larsen RS., 2003. Differential mortality of male spectacled eiders (Somateria fischeri) and king eiders (Somateria spectabilis) subsequent to anesthesia with propofol, bupivacaine, and ketoprofen. Journal of Avian Medicine and Surgery, 17, 117-123.
- Ninfali P., Orsenigo T., Barociani SR., 1990. Rapid purification of glucose 6-phosphogluconate dehydrogenase from mammal’s erythrocytes. Preparative Biochemistry and Biotechnology, 20, 297-309.
- Özabacıgil F., 2005. İnsan eritrosit 6-fosfoglukonat dehidrogenaz enziminin saflaştırılması, bazı ilaçların enzim aktivitesi üzerine in vitro ve tavşanlarda in vivo etkisinin incelenmesi. Atatürk Üniversitesi, Sağlık Bilimleri Enstitüsü, Erzurum, Türkiye.
- Ozabacigil F., Beydemir Ş., Çiftçi M., Gümüştekin K., Bakan N., 2008. Cisplatin and 5-fluorouracil inhibits 6-phosphogluconate dehydrogenase activity in human erythrocytes in vitro and in vivo. Asian Journal of Chemistry, 20, 3189- 3196.
- Puskas F., Gergely P., Banki K., Perl A., 2000. Stimulation of the pentose phosphate pathway and glutathione levels by dehydroascorbate, the oxidized form of vitamin C. The Journal of the Fedaration of American Societies for Experimental Biology, 14, 1352-1361.
- Segel IH., 1976. Biochemical calculations. John Wiley and Sons, Inc., 403, New York.
- Shreve DS., Levy HR., 1977. On the molecular weight of human glucose 6-phosphate dehydrogenase. Biochemical and Biophysical Research Communications, 78, 1369-1375.
- Tandoğan B., Ulusu N., 2003. Review. 6- Fosfoglukonat dehidrogenaz: Moleküler ve kinetik özellikler. Türk Biyokimya Dergisi, 28, 268-273.
- Walzem RL., Storebakken T., Hung SSO., Hansen RJ., 1991. Relationship between growth and selected liver enzyme activities of individual rainbow trout. Journal of Nutrition, 121, 1090- 1098.
- Williams RL., Upton RA., 1998. The clinical pharmacology of ketoprofen. Journal of Clinical Pharmacology, 28, 13-22.
Ayrıntılar
| Birincil Dil | Türkçe |
|---|---|
| Bölüm | Araştırma Makaleleri |
| Yazarlar | |
| Yayımlanma Tarihi | 23 Aralık 2014 |
| Yayımlandığı Sayı | Yıl 2014 Cilt: 9 Sayı: 3 |
