The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 μM. A change in matrix free Ca2+ from 0 to 0.3 μM caused a 135% increase in ADP stimulated oxidation of 0.6 mM α-ketoglutarate (K0.5 = 0.15 μM). In the absence of ADP and the presence of 0.6 mM α-ketoglutarate, Ca2+ (0.3 μM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 μM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess α-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 μM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 μM) than for α-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria α-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.
|Original language||English (US)|
|Number of pages||10|
|Journal||Journal of Biological Chemistry|
|State||Published - 1989|
All Science Journal Classification (ASJC) codes
- Molecular Biology
- Cell Biology