A Study of NADP~+- linked Isocitrate Dehydrogenase from Germinating Mung Bean (Vigna radiata)

摘要

NADP~+- linked isocitrate dehydrogenase has been purified to apparent homogeneity from 36 h germinated mung beans by ammonium sulphate fractionation, heat treatment, acid treatment, and DEAE - Cellulose column chromatography. The enzyme was purified to 150 fold with 15% recovery. The preparation showed single protein band on native PAGE and was free from bound nucleotides and coloured pigments (A_(280)/A_(260) = 1.4). The molecular weight was found to be 141,000 and was made of four identical subunits (mol wt 36,000). Thermal inactivation at 50, 53, and 55 °C revealed simple first order kinetics and t_(1/2) was found to be 38,10, and 3 min, respectively. The enzyme exhibited absolute specificity for NADP'and substrate. The K_m for isocitrate and NADP* was 28.57 pM and 70 pM, respectively. The enzyme appeared to be regulated by various metabolites of Krebs' cycle intermediates.