Disequilibrium in the malate dehydrogenase reaction in rat liver mitochondria in vivo.
1. When [2-(14)C]pyruvate is injected into rats the C3-position of liver glutamate becomes more heavily labelled than the C2-position, thus establishing that oxaloacetate and fumarate are not in equilibrium in rat liver mitochondria in vivo. The amount of disequilibrium was shown to be simply related to the value that the C3-label/C2-label ratio would have were no label recycled. This ratio, z, was calculated for post-absorptive rats in environmental temperatures of 20 degrees and 30 degrees C from determinations of the distribution of label within glutamate 1, 3 and 10min after intravenous injection of [2-(14)C]pyruvate. The values of z (best estimate and range) were 1.65 (1.60-1.69) in rats at 20 degrees C and 2.43 (2.23-2.63) in rats at 30 degrees C. These values of z imply the following rates of interconversion in mitochondria of fumarate and oxaloacetate (in terms of the oxaloacetate-->citrate flux, R) in rats at 20 degrees C: [Formula: see text] and in rats at 30 degrees C: [Formula: see text] 2. The kinetic parameters of malate dehydrogenase and fumarate hydratase and the intramitochondrial concentrations of NAD(+) and NADH under (as far as could be judged) conditions in vivo were collated. From them and the best estimates of R now available were calculated the rates of interconversion of fumarate, malate and oxaloacetate required to give the found values of z. These rates showed that the fumarate hydratase reaction was nearly in equilibrium, but that the malate dehydrogenase reaction was considerably out of equilibrium. The calculations also led to the following conclusions. 3. In livers of rats at 20 degrees and 30 degrees C mitochondrial malate concentrations were respectively about 5 and 1.5 times mean cellular concentrations. 4. Mitochondrial oxaloacetate concentrations were less than 0.2 of the mean cellular concentrations. They were also only 0.65 and 0.55 of the equilibrium concentrations for the malate dehydrogenase reaction in rats at 20 degrees and 30 degrees C respectively. 5. Malate dehydrogenase activity was low because of the very low oxaloacetate concentrations in the mitochondria and the very small fraction of the enzyme complexed with NAD(+), i.e. in each direction one substrate concentration was very sub-optimal.
D000818:Animals; D002247:Carbon Isotopes; D002951:Citrates / Q000378:metabolism; D005650:Fumarates / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D006836:Hydro-Lyases / Q000378:metabolism; D007700:Kinetics; D008291:Malate Dehydrogenase / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology* / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D010071:Oxaloacetates / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014867:Water / Q000378:metabolism
D002247:Carbon Isotopes; D002951:Citrates; D005650:Fumarates; D005971:Glutamates; D008293:Malates; D010071:Oxaloacetates; D011773:Pyruvates; D014867:Water; D009243:NAD; D008291:Malate Dehydrogenase; D006836:Hydro-Lyases