Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties.

Mustafa Erat

Biotechnology Application and Research Center, Atatürk University, Erzurum, Turkey. eratm@atauni.edu.tr

Preparative biochemistry & biotechnology 2005

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6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.

Citation

Mustafa Erat. Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties. Preparative biochemistry & biotechnology. 2005;35(1):53-69