Van Kedisi Eritrositi’nden 6-Fosfoglukonat Dehidrogenaz Enzimi (E.C. 1.1.1.44)’nin Saflaştırılması, Karakterizasyonu ve Kinetik Özellikleri

Bu çalışmada, 6-fosfoglukonat dehidrogenaz enzimi (6PGD; E.C.1.1.1.44) Van Kedisi eritrositinden afinite kromatografisi ile saflaştırıldı. Netilmicin sülfat, Ampicillin ve Amoxicillin’nin inhibitor etkisi incelendi. Enzim aktivitesi Beutler metoduna göre spektrofotometre ile 340 nm’de ölçüldü. Sonra enzimin optimum pH ve optimum sıcaklığı sırasıyla 8.0 ve 50 0C olarak tespit edildi. Enzimin saflığı sodyumdodesilsülfat- poliakrilamit jel elektroforezi (SDS-PAGE) ile kontrol edildi. 6PGD eritrositi için saflaştırma oranı 1787 olarak bulundu. NADP+ ve 6PGA substratları için KM ve Vmax değerleri hesaplandı. Netilmicin sülfat, Ampicillin ve Amoxicillin’nin inhibitor etkileri incelendi, Ki değerleri ve inhibisyon tipleri Lineweaver-Burk grafiğinden tespit edildi.

Anahtar Kelimeler:

Afinite kromatografisi, 6-fosfoglukonat dehidrogenaz enzimi, eritrosit, kinetik özellikler, Van Kedisi

Purification, Characterisation and Kinetic Properties of 6-Phosphogluconate Dehydrogenase Enzyme (E.C. 1.1.1.44) from the Van Cat Erythrocyte

In this study, 6-phosphogluconate dehydrogenase enzyme (6PGD; E.C.1.1.1.44) was purified from erythrocyte the Van Cat by affinity chromatography. The inhibitor effect of Netilmicin sulphate, Ampicillin and Amoxicillin were also examined. Enzyme activity was measured by spectrophotometer according to Beutler method at 340 nm. Then the optimum pH and optimum temperature of the enzyme were determined to be 8.0 and 50 ° C, respectively. In order to control the purification of enzyme, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used. The purification rate for erythrocyte 6PGD was found as 1787. KM and Vmax were also determined for NADP+ and 6PGA as substrates. The inhibitor effect of Netilmicin sulphate, Ampicillin and Amoxicillin were also examined and Ki values and the types of inhibition were determined by the Lineweaver-Burk graph.

Keywords:

Affinity chromatography, 6-Phosphogluconate dehydrogenase enzyme, erythrocyte, kinetic properties, Van Cat,

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1. Toews ML., Kanjı M., Carper W., 1976. 6-phosphogluconate dehydrogenase. The J of Biolog Chem, 251, 7127-7131. 2. Tandoğan B., Ulusu NN., 2003. 6-fosfoglukonat dehidrogenaz: Moleküler ve kinetik özellikleri. Türk Biyo Dergisi, 28, 268-273. 3. 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 Hemato, 80, 41-44. 4. Corrons V., Colomer D., Pujades A., Rovira A., Aymerich M., Merino A., 1996. Congenital 6-phosphogluconate dehydrogenase deficiency Associated with chronic hemolytic anemia in a Spanish family. American J of Hemato, 53, 221-227. 5. Walzem RL., Storebakken T., Hung SSO., Hansen RJ., 1991. Relationship between growth and selected liver enzyme activities of individual rainbow trout. J of Nutrition, 121, 1090-1098. 6. Barrett P., 1997. The pentose phosphate pathway and parasitic protozoa. Parasitol Today, 13 (1), 11-16. 7. Keha EE., Küfrevioğlu Öİ., 2000. Biyokimya. 344-345, Aktif Yayınevi, Ankara. 8. Gözükara E., 1989. Enzimler. ”Biyokimya”, 572-576, Ofset Repromat Ltd. Şti., Ankara. 9. Beutler E., 1971. Redcell metabolism. “Manual of biochemical methods”, 12, 68-70, Academic press, London. 10. Gür F., Beydemir Ş., Gümüştekin K., Bakan N., 2014. Ketoprofenin 6-fosfoglukonat dehidrogenaz aktivitesi üzerine in vitro ve in vivo etkisinin araştırılması. Atatürk Üni Vet Bil Derg, 9, 173-179. 11. Beydemir Ş., Çiftçi M., Yılmaz H., Küfrevioğlu OI., 2004. 6-phosphogluconate dehydrogenase: Purification, characterization and kinetic properties from Rat erythrocytes. Turk J of Vet Ani Sci, 28, 707-714. 12. Laemmli DK., 1970. Clevage of structural proteins during in assembly of the head of bacteriophage T4. Nature, 227, 680-683. 13. Bradford MM., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analy Biochem, 7, 248-254. 14. Özabacıgil F., 2005. İnsan eritrosit 6-fosfoglukonat dehidrogenaz enziminin saflaştırılması, bazı ilaçların enzim aktivitesi üzerine in vitro ve tavşanlarda in vivo etkisinin incelenmesi. Atatürk Üni, Sağ Bil Ens, Erzurum, Türkiye. 15. Akyüz M., Erat M., Çiftçi M., Gümüştekin K., Bakan N., 2004. Effects of some antibiotics on human erythrocyte 6-Phospho Gluconate Dehydrogenase: An in vitro and in Vivo study. J of Enzyme Inhi and Medicinal Chem, 19, 361-365. 16. Adem Ş., Çiftçi M., 2007. Effects of some drugs on human erythrocyte 6-phosphogluconate dehydrogenase: an in vitro study. J of Enzyme Inhi and Med Chem, 22, 751-754. 17. Ciftci M., Beydemir S., Yılmaz H., Bakan E., 2002. Effects of some drugs on rat erythrocyte 6-phosphogluconate dehydrogenase: An in vitro and in vivo study. J Pharma, 54:275-280. 18. Weisz KS., Schofield PJ., Edwards MR., 1985. Human brain 6-PGD: Purification and kinetic properties. J of Neurochem, 44, 510-517. 19. Demir H., Çiftçi M., Küfrevioğlu Öİ., 2003. Purification of 6-phosphogluconate dehydrogenase from Parsley (Petroselinum hortense) leaves and investigation of some kinetic properties. Prep Biochemhem and Biotech ,33, 39-52. 20. Krepinsky K., Plaumann M., Martin W., Schnarrenberger C., 2001. Purification and cloning of chloroplast 6-PGD from spinach. Euro J of Biochem, 268, 2678-2686. 21. Ceyhan D., Danısan A., Oğu, IH., Ozer N., 2005. Purification and kinetic properties of 6-phosphogluconate dehydrogenase from rat small intesine. The Protein J, 24, 293-301. 22. Ohara H., Russell RA., Uchide K., Kondo H., 2004. Purification and characterization of NAD spesific 6-phosphogluconate dehydrogenase from Leuconostoc lactis SHO-54. J of Biosci and Bioengin, 98, 126-128. 23. Erat M., 2005. Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of same kinetic properties. Prep Biochemhem and Biotechno, 35, 53-69.