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Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Purificación y caracterización de la glucosa-6-fosfato deshidrogenasa de hígado del pezChalcalburnus tarichii

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Abstract

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2′, 5′-ADP Sepharose 4B affinity gel chromatography, which took 7–8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1,310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6- phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.

Resumen

Mediante un método simple y rápido, se identifica la enzima glucosa-6-fosfato deshidrogenasa (D-glucosa-6-fosfato: NADP+óxidorreductasa, EC 1.1.1.49, G6PD) a partir de hígado del pezChalcalburnus tarichii y se investigan sus características. El procedimiento de purificación incluye 2 fases, preparación del homogenado y cromatografía en gel de afinidad con 2′, 5′-ADP sefarosa 4B, lo que supone 7–8 horas. Tras los 2 procedimientos consecutivos, se logra purificación de la enzima, de actividad específica 36,26 EU/ mg de proteína, con un 44,48% de rendimiento y un enriquecimiento de 1.215. Para controlar la purificación se realizó SDS-PAGE. Se han determinado los valores de pH óptimo y estable, de la temperatura óptima, de Km y de Vmax para NADP+ y glucosa-6-fosfato. Además, se han estimado los valores del peso molecular de la enzima y del de cada subunidad medianteSDS-PAGE y cromatografía de filtración en gel respectivamente.

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Authors and Affiliations

  1. Arts and Science Faculty, Department of Chemistry, 100. Yil University, Van

    V. Turkoglu & M. Altun

  2. Arts and Science Faculty, Department of Chemistry, Ataturk University, 25240, Erzurum, Turkey

    M. Çiftçi

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  1. V. Turkoglu
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  2. M. Altun
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  3. M. Çiftçi
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Correspondence to M. Çiftçi.

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Turkoglu, V., Altun, M. & Çiftçi, M. Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver. J. Physiol. Biochem. 62, 155–161 (2006). https://doi.org/10.1007/BF03168464

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  • DOI: https://doi.org/10.1007/BF03168464

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