Purification of 6-phosphogluconate dehydrogenase from parsley (Petroselinum hortense) leaves and investigation of some kinetic properties.
In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)(-1) (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558-560). The overall purification was about 339-fold. A temperature of +4 degrees C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 mn. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50 degrees C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25 degrees C were determined by means of Lineweaver-Burk plots.
Demir
Hülya
H
Biotechnology Application and Research Center, Atatürk University, Erzurum, Turkey.
Ciftçi
Mehmet
M
Küfrevioğlu
O Irfan
OI
D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D028528:Petroselinum / Q000737:chemistry* / Q000201:enzymology; D010734:Phosphogluconate Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification*; D018515:Plant Leaves / Q000737:chemistry* / Q000201:enzymology; D013696:Temperature
D010734:Phosphogluconate Dehydrogenase; C029757:phosphogluconate dehydrogenase (decarboxylating)