Purification and Properties of Diphosphopyridine Nucleotide-linked Isocitrate Dehydrogenase of Mammalian Liver

DPN-linked isocitrate dehydrogenase has been purified approximately 100-fold from extracts of porcine liver mitochondria. The purified enzyme is free of TPN-linked isocitrate dehydrogenase, glutamate dehydrogenase, aconitase, and ATP-AMP transphosphorylase activity. ADP, 5'-AMP, and cyclic 3',5'-AMP are positive modifiers of the enzyme above pH 6.5; ADP is a better activator than the mononucleotides. The enzyme has optimal activity at pH 6.5 with low isocitrate concentration and without ADP; with ADP (0.5 to 1.5 mm) the optimum is at pH 7.3, with high or low concentrations of isocitrate. The interaction of isocitrate and DPN+ is that of a typical two-substrate system. At pH 7.2 with 1.3 mm Mn++, the limiting Michaelis constants of threo-d-isocitrate are 0.32 mm and 0.26 mm, and those of DPN+ are 0.06 mm and 0.09 mm, in the absence and presence of 0.67 mm ADP, respectively. While Kdpn, Kisocitrate, and Vmax are essentially unchanged by the presence of ADP, the ratio of complex constants, φ12/φ1 and φ12/φ2, are 4- to 7-fold larger in the absence than in the presence of ADP. The apparent Km of Mg++ is lowered by increased isocitrate concentration; ADP further decreases the value of this constant. DPNH and ATP inhibit activity competitively with DPN+, and TPNH synergistically enhances the inhibition by DPNH. The Ki of DPNH (0.016 mm) is essentially uninfluenced by the concentration of isocitrate or by ADP. The inhibition by 67 µm p-chloromercuribenzenesulfonate can be partially prevented by the presence of isocitrate and Mn++; however, the addition of DPN+ and all other medium components in the absence of isocitrate does not afford such protection. At pH 7.2 and with 1.3 mm Mn++, Michaelis kinetics has been observed over a wide range of concentrations of isocitrate and DPN+, with or without ADP. Inhibition by DPNH or ATP does not induce cooperative effects of substrates under these conditions. The possible regulatory role of various factors influencing DPN-linked isocitrate dehydrogenase activity in the metabolism of liver mitochondria is discussed.