Activity of purified NAD-specific isocitrate dehydrogenase at modulator and substrate concentrations approximating conditions in mitochondria

The kinetic parameters of NAD-specific isocitrate dehydrogenase from bovine heart were examined at levels of substrates and effectors approximating the concentrations reported for isolated intact heart mitochondria in different respiratory states. The effect of changing ADP/ATP ratios (with total adenine nucleotides constant at 8 mmol/L) on enzyme activity was measured at constant concentrations of the substrates magnesium D-isocitrate (0.10 mmol/L) and NAD+ (3.0 mmol/L), the positive effector magnesium citrate (1.0 mmol/L) and the negative effector NADPH (1.5 mmol/L) at pH 7.4. Enzyme activity increased with increasing ADP/ATP ratios as a result of activation by rising ADP concentrations and not due to decreasing inhibition by falling levels of ATP. Increasing ADP decreased the inhibition by NADPH, and this effect was enhanced by magnesium citrate and by free Ca2+. In incubation media containing all of the above effectors, the S0.5 for enhancement of activity by free Ca2+ was 10 to 20 mumol/L at ratios of total ADP/total ATP between 2.0 and 0.1. This value is in the range of intramitochondrial concentrations of free Ca2+,1 but it is appreciably larger than S0.5 of Ca2+ (0.6 to 1 mumol/L) for the enhancement of ADP activation, which was determined in the absence of other effectors. When both the NAD+/NADH and the ADP/ATP ratios were decreased, a further decline in activity was found. The effect of the decreasing NAD+/NADH ratio was due to inhibition by NADH (apparent I0.5 = 0.23 +/- 0.03 mmol/L) since NAD+ was saturating over the range examined.