Purification and characterization of 6-phosphogluconate dehydrogenase (6-PGD) from grass carp (<i style="mso-bidi-font-style: normal">Ctenopharyngodon idella</i>) hepatopancreas

Abstract

6-Phosphogluconate dehydrogenase (6-PGD, E.C.: 1.1.1.44) was purified and characterized from the hepatopancreas of grass carp (<i style="mso-bidi-font-style:normal">Ctenopharyngodon idella</i>) for the first time. Grass carp represents the second largest aquaculture industry in the world after silver carp, constituting 14.7% of the world aquaculture production, with an average annual increase of 14% in China, mainly as a source of food. The purification procedure involved a single 2’, 5’-ADP-Sepharose 4B affinity chromatographic step by using different elution buffers. The enzyme was purified 309-fold with a specific activity of 5.259 U/mg protein and yield of 68%. The purity and subunit molecular weights of the 6-PGD were checked on SDS-PAGE and purified enzyme showed a single band on the gel. The subunit molecular mass was 57 kDa, with an optimum pH, temperature and ionic strength at 7.96, 50^oC and 100 mM Tris-HCl, respectively. The <i style="mso-bidi-font-style:normal">K_m</i> values of 6-PGA and NADP⁺were 0.019 and 0.0052 mM, respectively, while<i style="mso-bidi-font-style:normal"> V_m </i>of 6-PGA and NADP⁺ was 0.69 U/ml. Dissociation constants (<i style="mso-bidi-font-style:normal">K_i</i>) for 6-PGA and NADP⁺were 2.05 and 0.12 mM, respectively. NADPH inhibited the enzyme in a competitive manner and its<i style="mso-bidi-font-style:normal"> K_i</i> value was 0.032 mM. The Cu²⁺, Zn²⁺, Cd²⁺ and Al³⁺ showed inhibitory effects on the enzyme with IC₅₀ values of 0.293, 0.099, 0.045 and 1.526 mM, respectively. All tested metals inhibited the enzyme in a competitive manner, indicating that these metals might be toxic even at low concentrations for the 6-PGD. As the fish is one of valuable foodstuff of animal sources for human consumption, under certain environmental conditions, metal ions accumulated in fish up to a lethal concentration may be harmful for human health. Therefore, it is impending to reduce the concentration of metal ions in contaminated lakes and rivers for fishery and also for human health.