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

Yıl 2017, Cilt: 12 Sayı: 2, 187 - 194, 30.10.2017

Serpil Kılıç

https://doi.org/10.17094/ataunivbd.347975

Öz

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 K_M 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

Öz

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. K_M and V_max
were also determined for NADP⁺ and 6PGA as substrates. The inhibitor
effect of Netilmicin sulphate, Ampicillin and Amoxicillin were also examined and K_i
values and the types of inhibition were determined by the Lineweaver-Burk
graph. 

Anahtar Kelimeler

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

Kaynakça

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