Şükrü BEYDEMİR, Ö. İrfan KÜFREVİOĞLU, Mehmet ÇİFTÇİ, Hayrullah YILMAZ

6-Phosphogiluconate dehydrogenase: Purification, characterization and kinetic properties from rat erythrocytes

Bu çalışmada 6-fosfoglukonat dehidrogenaz'ın sıçan eritrositlerinden saflaştırılması için basit ve hızlı bir metod ile birlikte bu enzimin kinetik davranışı ve özelliklerinin analizi çalışılmıştır. Saflaştırma basamakları yüksek hızda santrifügasyon, % 20-50 amonyum sülfat çöktürmesi ve 2', 5'-ADP Sepharose 4B afinite jel kromatografısini içerdi. Verim % 78,4 oldu ve enzimin spesifik aktivitesi 5,15 EU/mg protein olarak bulundu. Altbirim molekül kütlesi SDS-poliakrilamid jel elektroforezi (SDS-PAGE) vasıtasıyla 59,566 Da olarak hesaplandı ve enzimin tabii halinin molekül kütlesi jel filtrasyon kolon kromatografisi ile 111,000 Da olarak bulundu. Enzim 1 M Tris-HCl tampon çözeltisi içinde pH: 7,0'da optimum, pH: 8,0'da stabil pH'ya ve 45 °C'de optimum sıcaklığa sahipti. Aynı zamanda $NADP^+$ ve 6-PGA substratları için $K_M ve V_{max}$ değerleri belirlendi. ATP, NADPH ve NADH'ın inhibitor etkileri incelendi. $K_İ$ değerleri ve inhibisyon tipleri bu inhibitörler için belirlenen Lineweaver-Burk grafikleriyle saptandı.

6-Fosfoglukonat dehitrogenaz: Sıçan eritrositlerinden saflaştırılması, karakterizasyonu ve kinektik özellikleri

In this paper, a simple and rapid method for the purification of 6-phosphogluconate dehydrogenase from rat erythrocytes together with an analysis of the kinetic behavior and some properties of the enzyme are considered. The purification steps comprised high-speed centrifugation, 20-50% ammonium sulfate precipitation and 2', 5'-ADP Sepharose 4B affinity gel chromatography. The yield was 78.4% and the specific enzyme activity was 5.15 EU/mg proteins. The molecular mass of the subunit was estimated to be 59,566 Da by SDS polyacrylamide gel electrophoresis (SOS-PAGE) and native enzyme was found to be 111,000 Da by gel filtration column chromatography. The enzyme had an optimal pH at 7.0 and stable pH at 8.0 in 1 M Tris-HCl buffer, and optimal temperature at 45 °C. $K_M and V_{max}$ for $NADP^+$ and 6-PGA as substrates were also determined. The inhibitor effects of ATP, NADPH and NADH were also examined, and $K_İ$ values and the types of inhibition were determined by means of a Lineweaver-Burk graph obtained for them.

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