Purification of 6-Phosphogluconate Dehydrogenase from Parsley (Petroselinum hortense) Leaves and Investigation of Some Kinetic Properties

Abstract

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.

Purification and Characterization of Glucose 6‐Phosphate Dehydrogenase, 6‐Phosphogluconate Dehydrogenase, and Glutathione Reductase from Rat Heart and Inhibition Effects of Furosemide, Digoxin, and Dopamine on the Enzymes Activities

“…We purified successfully three enzymes from rat heart using the affinity chromatography method. In some reports, the purification of these enzymes has been severally performed using multistep chromatographic methods including affinity, ion‐exchange, and gel filtration chromatography . 2′,5′‐ADP sepharose‐4B affinity chromatography is a special method for purification of NADP + /NADPH‐dependent enzymes such as 6PGD, GR, and G6PD.…”

Purification and Characterization of 6-Phosphogluconate Dehydrogenase from Japanese Quail (Coturnix, coturnix japonica) Erythrocytes

“…(Coturnix coturnix japonica) erythrocytes for the first time. Different methods were used for the purification of 6PGD enzyme throughout the literature of enzymology like DEAE cellulose; CM-cellulose ions exchange chromatography (Villet and Dalziel, 1969), DEAE Sephadex, hydroxy apatite (Silverberg and Dalziel, 1973), NADP + Sepharose, NADP + agarose (Betts and Mayer, 1975), matrix gel-A column chromatography (Somers et al 1991), DEAE Sephadex A-50 ion-exchange chromatography (Demir et al, 2003), 2', 5'-ADP-sepharose 4B affinity gel (Altıkat et al, 2002;Akyüz et al, 2003;Bayındır et al, 2018;Akkoyun et al, 2018). The method used for the purification of the enzyme was 2ˈ, 5ˈ-ADP Sepharose 4B affinity gel chromatography, which is a very powerful, reliable, easy, economic and a single step purification method that gives very good purification yields with an ability to purify bulk amounts of the enzymes (Temel et al, 2017a,b;Temel and Kocyigit, 2017).…”

“…6PGD enzyme had been investigated in many different sources and checked for its kinetic properties and characterized for its functions. The enzyme had been purified from sheep's liver (Villet and Dalziel, 1972), parsley's leaves (Demir et al, 2003), chicken's liver (Erat, 2005), rat's small intestine (Ceyhan et al, 2005), Van's cat erythrocytes (Kiliç, 2007), human's erythrocytes (Özabacigil, 2005), human cerebral (Weisz et al, 1985), rabbit mammary glands (Betts and Mayer, 1975) and rat heart and lung tissues (Adem, 2010). The enzyme's molecular weight was determined by SDS-PAGE method described previously.…”

“…The molecular weight of the subunit of the purified enzyme found to be ~81 kDa. The molecular weights of 6PGD enzymes from different sources in literature had been 59.5 kDa from the rat's erythrocytes (Beydemir et al, 2004), 52.6 kDa from Van's cat erythrocyte (Kiliç, 2007), 52 kDa from rat's small intestine (Ceyhan et al, 2005), E. coli bacteria had 53 kDa 6PGD (Wang and Zhang, 2009), 118.34 kDa from rainbow trout by gel filtration chromatography (Taranci, 2011) and 24.1 kDa from parsley (Demir et al, 2003). By looking through above molecular weights from different sources, we find that the molecular weight of 6PGD enzyme from Japanese quail's is different from all the mentioned sources.…”

Development of a fluorescence-based cellular apoptosis reporter

“…Addition of the cleavage domain did not affect the cellular distribution of GFP (figure 1(B)). We also generated non-cleavable reporters in which the aspartic acid at the P 1 position was changed to an asparagine residue yielding the control constructs NGN6 and NGNH ([22–24], figure 1(A)).…”