Isocitrate dehydrogenases from liver ofSqualus acanthias (spiny dogfish) and citrate formation by isolated mitochondria

Isolated mitochondria from liver of Squalus acanthias (spiny dogfish) catalyze formation of citrate from α-ketoglutarate at a rate corresponding to >0.03 μmoles per min per g tissue. Malate alone does not support citrate formation, but stimulates citrated formation from α-ketoglutarate ≈︂ threefold. These reactions appear to proceed by reductive carboxylation catalyzed by the mitochondrial NADP-dependent isocitrate dehydrogenase, i.e., reversal of the Krebs cycle from α-ketoglutarate to citrate. The NADP-dependent isocitrate dehydrogenases present in the mitochondria and cytosol were isolated and shown to have different N-terminal amino acid sequences, indicating that they are encoded by different genes. The kinetic and structural properties of the mitochondrial enzyme were found to be similar to that of NADP-dependent isocitrate dehydrogenases from other species. NAD-dependent isocitrate dehydrogenase activity could not be detected in liver extracts or in mitochondrial or cytosolic subreactions of liver extracts. The level of activity of the reverse reaction (≈︂0.4 μmoles per min per g liver), i.e., reductive carboxylation, of the mitochondrial isocitrate dehydrogenase is sufficient to account for the observed rate of citrate formation by isolated mitochondria. Isolated mitochondria also catalyze citrate formation from pyruvate plus malate at a rate that is ≈︂70% of the rate observed with α-ketoglutarate plus malate. The levels of ATP citrate lyase (shown to be present in the cytosol) and cytosolic NADP-dependent isocitrate dehydrogenase activities (≈︂0.5 and 2.5 μmoles per min per g liver, respectively) are sufficient to utilize the observed optimal rate of mitochondrial citrate formation from α-ketoglutarate and pyruvate in the presence of malate (≈︂0.12 μmoles per min per g liver) for acetyl-CoA and NADPH formation required for lipid biosynthesis and-or for oxalacetate formation required for gluconeogensis.