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Effects of micromolar concentrations of free calcium ions on the reduction of heart mitochondrial NAD(P) by 2-oxoglutarate.

ABSTRACT: 1. The reduction of mitochondrial NAD(P) by 2-oxoglutarate was monitored as a measure of 2-oxoglutarate dehydrogenase activity in its intramitochondrial locale. In the absence of ADP, steady-state reduction of NAD(P) by 0.5 mM-2-oxoglutarate in the presence of 0.5 mM-L-malate was markedly increased by extramitochondrial Ca2+, with 50% activation at pCa 6.58, when the Na+ concentration was 10 mM, the Pi concentration ws 5 mM and the added Mg2+ concentration was 1 mM. Omission of Pi resulted in 50% activation at pCa 6.77; omission of Mg2+ resulted in 50% activation at pCA greater than or equal to 7.3. 2. The activation of 2-oxoglutarate dehydrogenase could be reversed on addition of an excess of EGTA. The rate of inactivation was dependent on the concentration of Na+, with K0.5 2.5 mM, which is consistent with the rate of withdrawal of Ca2+ from the mitochondria being the limiting factor. 3. The steady-state reduction of cytochrome c by 2-oxoglutarate (0.5 mM) also showed a marked dependence on pCa in the absence of ADP; in the presence of an excess of ADP, no such effect of Ca2+ was detectable. 4. Mitochondria from the hearts of senescent rats showed an undiminished rate of dehydrogenase activation by Ca2+ but a rate of inactivation by excess EGTA that was diminished by 40%. Direct studies of Ca2+ egress with Arsenazo III confirmed a decrement in rate with old age. 5. Studies of 2-oxoglutarate dehydrogenase activity as a function of the mitochondrial context of Ca2+, as measured by atomic-absorption spectrophotometry, showed half-maximal activation at a mitochondrial content of 1.0 nmol of Ca2+/mg of protein, and saturation at 3 nmol/mg. 6. These findings support the model advanced by Denton, Richards & Chin [(1978) Biochem. J. 176, 899-906], of a control of the tricarboxylate cycle by intramitochondrial Ca2+, and demonstrate the range of mitochondrial Ca2+ content over which this may occur. In addition, they raise the possibility of a disturbance of this control mechanism in old age.

SUBMITTER: Hansford RG

PROVIDER: S-EPMC1163298 | biostudies-other | 1981 Sep

REPOSITORIES: biostudies-other

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Project description:1. The mitochondrial content of active (dephospho) pyruvate dehydrogenase (PDHA) was found to be severalfold higher at an extramitochondrial Ca2+ concentration of 2 microM (pCa6) than at pCa7. The nature of the respiratory substrate did not affect this finding. 2. This Ca2+-dependence was shown in state-4 and 50%-state-3 conditions [see Chance & Williams (1956) Adv. Enzymol. 17, 65-134], but was absent in the presence of excess ADP (state 3). 3. Na+ and Mg2+ ions shifted the pCa value required for a maximal PDHA content to lower values. This was attributed to a stimulation of mitochondrial Ca2+ egress and an inhibition of uptake, respectively. Na+ ions diminished pyruvate dehydrogenase phosphate phosphatase activity in mitochondria which had been extensively depleted of Ca2+ ions by incubation with EGTA, raising the possibility of a direct inhibitory effect of Na+ ions, unrelated to Ca2+ movements. 4. Mg2+ ions lowered the mitochondrial PDHA content at pCa 6.24 and 6.48, but had only minimal effects in the presence of EGTA. 5. The effects of P1 and bicarbonate ions on PDHA content were also studied, as possible effectors of mitochondrial Ca2+ transport. Bicarbonate ions abolished the response to Ca2+ ions, by generating maximal values of PDHA content, but such a response was still observed when physiological concentrations of both P1 and bicarbonate were used. 6. The pCa of the medium in the range 6.33 to over 7 affected PDHA content, with only very minor changes in state-4 rates of O2 uptake and no change in [ATP]/[ADP] ratio or in mitochondrial [NADH]/[NAD+] ratio, provided that Mg2+ ions were present. Thus the effect of Ca2+ ions on PDHA content is unlikely to be mediated by changes in [ATP]/[ADP] and [NADH]/[NAD+] ratio and is more likely to be direct. Equally, changes in the [acetyl-CoA]/[CoA] ratio in response to Ca2+ ions when the substrate was pyruvate were the converse of those required to mediate changes in interconversion, and are probably secondary to changes in PDHA content.

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Effects of micromolar concentrations of free calcium ions on the reduction of heart mitochondrial NAD(P) by 2-oxoglutarate.

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