Isolation and Characterization of NADP+-Linked Isocitrate Dehydrogenase in Germinating Urd Bean Seeds (Phaseolus mungo)

摘要

Isocitrate dehydrogenase (EC 1.1.1.42) has been purified to homogeneity from germinating urd bean seeds. The enzyme NADP+-linked isocitrate dehydrogenase is a tetrameric protein (molecular weight 130,000; gel filtration) made up of four identical monomers (subunit molecular weight about 32,000-33,000; PAGE in presence of sodium dodecyl sulphate). Thermal inactivation of purified enzyme at40 °C, 45 °C and 50 °C shows single exponential loss of enzyme activity suggesting that the inactivation of this enzyme follows simple firstorder kinetics (rate constants for purified enzyme 0.020, 0.043 and 0.077 min–1 at 40 °C, 45 °C and 50 °C respectively). Thermal inactivationin presence of glutathione and dithiothretol at 45 °C and 50 °C also follows simple first order kinetics, but the presence of these compoundsprotects the loss of enzyme activity. The enzyme shows optimum activity at pH 7.3-8.0. The variation of Vmax and Km at different pH values(6.5-8.0) suggests that proton behaves as an "Uncompetitive Inhibitor". A basic group is present at the active site of enzyme which isaccessible for protonation in this pH range in the presence of substrate only, with a pKa equal to 6.8. Successive dialysis against EDTA andphosphate buffer, pH 7.5 at 0-4 °C gives an enzymatically inactive protein. Thermal inactivation of this protein at 45 °C and 50 °C shows anexponential loss of enzyme activity as in the case of untreated (native) enzyme. Full activity is restored on adding Mn2+ (3.75mM) to asolution of this protein. Addition of Mg2+, Zn2+, Co2+ and Cu2+ brings about partial recovery. Alkali metal ions bring about 75% inhibitionat 4mM concentration. The inhibition is stronger at high concentration of Na+ and K+. Other metal ions are not effective.