The activity of pyruvate carboxylase in isolated, tightly coupled, rat liver mitochondria was estimated by measuring the incorporation of H14CO3 into acid-stable metabolites in the presence of pyruvate and ATP. The addition of a given amount of Ca2+ (10 to 100 nmol/mg of protein) to isolated mitochondria catalyzing the incorporation of H14CO3- into acid-stable intermediates at a steady state rate caused a rapid decrease in the rate of H14CO3- incorporation, and the establishment of a new inhibited steady state rate. This effect corresponded with the uptake of Ca2+ (monitored with the use of 45Ca) from the incubation medium by the mitochondria. When the steady state rate of H14CO3- incorporation was measured in the presence of various fixed concentrations of added Ca2+, it was found to decrease as the concentration of intramitochondrial Ca2+ increased from 21 (no added Ca2+) to 120 nmol/mg of protein (100 nmol/mg of added Ca2+). Inhibition by Ca2+ was more pronounced at low concentrations of pyruvate. Thus a 50% inhibition of the rate of H14CO3- incorporation was observed after the addition of either 25 or 10 nmol/mg of exogenous Ca2+ at 7.5 and 1 mM pyruvate, respectively. Inhibition of H14CO3- incorporation by Ca2+ could be completely reversed by the subsequent addition of Mn2+ (25 to 100 nmol/mg). An increase in intramitochondrial Ca2+ (from 20 to 50 nmol/mg of protein) was accompanied by 25% increase in the activity of active form of pyruvate dehydrogenase. Elevated concentrations of intramitochondrial Ca2+ (50 nmol/mg of exogenous Ca2+) were associated with (a) an 80% inhibition of the rate of mitochondrial ATP transport and a decrease of 50% in the pool of intramitochondrial adenine nucleotides available for exchange with exogenous [14C]ATP (measured at 0°); (b) a 40% inhibition of the rate of mitochondrial pyruvate transport measured at 6° in the presence of rotenone and antimycin A; (c) a 20% decrease in the intramitochondrial pyruvate concentration; (d) a small increase in the intramitochondrial concentration of acetyl-CoA; and (e) no significant change in the intramitochondrial concentrations of total ATP and ADP. A control experiment showed that an increase in intramitochondrial Ca2+ from 20 to 65 nmol/mg of protein caused a small stimulation of the activity of the citric acid cycle when this was estimated by measuring the release of 14CO2 in the presence of [U-14C]malate. It is concluded that (a) inhibition by Ca2+ of H14CO3- incorporation is the result of inhibition of the pyruvate carboxylase enzyme by CaATP2+ and Ca2+ present in the mitochondrial matrix as well as a decrease in the concentration of intramitochondrial pyruvate; (b) the data are consistent with the proposal that changes in the intramitochondrial concentrations of Ca2+ can contribute to the regulation of the activity of pyruvate carboxylase in the liver; and (c) under some conditions, changes in the mitochondrial Ca2+ concentrations may regulate cellular metabolism through their effects on mitochondrial ATP and pyruvate transport.
|Number of pages||9|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - 10 Aug 1977|