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

Purification of glucose 6-phosphate dehydrogenase from goose erytrocytes and kinetic properties

Glukoz 6-fosfat dehidrogenaz (G6PD) enzimi kaz eritrositlerinden saflaştırıldı ve bazı karakteristik özellikleri araştırıldı. Saflaştırma prosedürü;' hemolizatın hazırlanması, amonyum sülfat çöktürmesi ve 2', 5'-ADP Sepharose 4B afınite kromatografisi şeklinde üç basamaktan ibarettir. Bu üç basamak neticesinde 36,2 EÜ/mg protein spesifik aktivitesine sahip olan enzim, % 68,79 verimle 3.892 kat saflaştırıldı ve enzim saflığının kontrol edilmesi için SDS-PAGE yapıldı. Enzim için optimum pH, stabil pH, optimum sıcaklık, molekül ağırlığı, NADP* için Km ve Vmax değerleri, G6-P için Km ve Vmax değerleri hesaplandı. Ayrıca ATP, ADP ve NADPH inhibitörleri için Lineweaver-Burk grafikleri çizilerek K, değerleri ve inhibisyon tipleri tespit edildi. Bu maddeler enzimi yarışmasız olarak inhibe etti.

Kaz eritrositlerinden glukoz 6-fosfat dehidrogenaz enziminin saflaştırılması ve kinetik özellikleri

Glucose 6-phosphate dehydrogenase (G6PD) was purified from goose erythrocytes and some characteristics of the enzyme were investigated. The purification procedure was composed of 3 steps: hemolysate preparation, ammonium sulfate precipitation, and 2', 5'-ADP Sepharose 4B affinity gel chromatography. Thanks to the 3 consecutive procedures, the enzyme, having a specific activity of 36.2 EU/mg protein, was purified for a yield of 68.79% and 3892 folds; to ascertain enzyme purity, SDS-PAGE was performed. Optimal pH, stable pH, optimal temperature, molecular weight, and Km and Vmax values for NADP* and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. In addition, K, values and inhibition type were determined by means of Lineweaver-Burk graphs obtained for such inhibitors as ATP, ADP and NADPH. These materials inhibited the enzyme in a noncompetitive manner.

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