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AI InsightThe inhibitor effects of ATP, NADPH and NADH were also examined, and Ki values and the types of inhibition were determined by means of a Lineweaver-Burk graph obtained for them.
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Özet
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 (SDS-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 ºC. KM and Vmax for NADP+ and 6-PGA as substrates were also determined. The inhibitor effects of ATP, NADPH and NADH were also examined, and Ki values and the types of inhibition were determined by means of a Lineweaver-Burk graph obtained for them.
Abstract
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 (SDS-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 ºC. KM and Vmax for NADP+ and 6-PGA as substrates were also determined. The inhibitor effects of ATP, NADPH and NADH were also examined, and Ki values and the types of inhibition were determined by means of a Lineweaver-Burk graph obtained for them.