DPN-linked isocitrate dehydrogenase has been purified from mitochondrial acetone powder of bovine heart. The purified protein exhibits a major component having a sedimentation constant of 10.3 S in the analytical ultracentrifuge, with a possible molecular weight in the range 3 4 X 10s. ADP has been found to affect the enzyme in several ways. The nucleotide stabilizes the enzyme under conditions of low ionic strength. ADP also enhances the activity of the enzyme, and this effect has been found to be due chiefly to a marked decrease in the K,,, for isocitrate. Thus, a t low isocitrate concentrations the enzyme is virtually dependent on ADP for activity. This effect of ADP is highly specific, since, of a large number of nucleotides tested, only ADP and dADP are stimulatory. In the presence of low concentrations of isocitrate, the p H optimum is displaced from p H 6.7 in the absence of ADP to about pH 7.2 in the presence of ADP. A similar shift of the pH optimum is obtained by increasing the substrate concentration in the absence of ADP. The enzyme is inhibited by DPNH, ATP, and ADPR; the inhibition is competitive with DPN+. TPNH potentiates the DPNH inhibition of the DPN-linked isocitrate dehydrogenase. On the other hand, the initial reaction rate of the TPN-linked isocitrate dehydrogenase system is not affected by ADP, ATP, DPN +, or DPNH. In the presence of DPN-linked isocitrate dehydrogenase, the fluorescence emission spectrum of DPNH is altered, suggesting the formation of a DPNH-enzyme complex. The significance of the inhibition by ATP and DPNH, as well as the ADP stimulation, is discussed in terms of a possible mechanism for regulation of cardiac mitochondrial oxidation.It has been shown (Plaut and Sung, 1954) that a number of animal tissues contain a DPN-linked isocitrate dehydrogenase catalyzing the reaction threo-D,-isocitratel + D P N + --+ a-ketoglutarate + CO, + DPNH + H + A preliminary communication (Chen and Plaut, 1962) reported that enzyme from bovine heart was markedly stimulated by ADP and that this nucleotide markedly decreased the apparent Michaelis constant K, for isocitrate. The discovery of the activating effect of ADP on this enzyme occurred during the course of a systematic survey of the effect of various nucleotides. This study was undertaken for several reasons. First, Kornberg and Pricer (19511, who initially described a DPN-linked kcitrate dehydrogenase, found that the yeast enzyme was almost fully dependent on minute quantities of 5'-AMP, although ADP in larger amounts could also activate the enzyme. In Aspergillus niger, DPN-linked isocitrate dehydrogenase has been reported to be activated by 5'-AMP and by inorganic phosphate ( h a k r i s h n a n and Martin, 1955). Although 5'-AMP had no such effect on the mammalian DPN-specific isocitrate dehydrogenase (Plaut and Sung, 1954), it remained possible that the enzyme might be activated by other nucleotides. Second, the DPNlinked enzyme of cardiac muscle had been observed to be inhibited by ATP (Plaut and Sung, 1954). Also the rate of isoc...

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Cryptates, a new type of metal cation complexes, have been described recently.12 It was found that the

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