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Moreno-Sanchez R, Hansford RG: Dependence of cardiac mitochondrial pyruvate dehydrogenase activity on intramitochondrial free Ca2+ concentration. Biochem J. 1988 Dec 1;256(2):403-12.
(1) The free Ca2+ concentration of the matrix of rat heart mitochondria ([Ca2+] m) was determined from the fluorescence of internalized indo-1. The value of the Kd of indo-1-Ca2+ in the mitochondrial matrix was determined to be 95 nM, on the basis of equilibration of [Ca2+] m with the extramitochondrial free Ca2+ ([Ca2+] o) in the presence of rotenone, nigericin, valinomycin and Br-A23187. (2) [Ca2+] m responded to energization/de-energization protocols, the inhibition of Ca2+-uptake by Ruthenium Red and the potentiation of Ca2+-efflux by Na+ in a manner which was consistent with the known kinetic properties of the mitochondrial Ca2+-transport processes. (3) The concentration gradient [Ca2+] m/[Ca2+] o was found to be near unity (0.82 +/- 0.18) when mitochondria were incubated in media containing 10 mM-Na+; the additional presence of 1 mM-Mg2+ reduced the gradient to values below unity (0.26 +/- 0.03). The polyamine spermine increased the Ca2+ concentration gradient in the presence of 1 mM-Mg2+. (4) The fraction of pyruvate dehydrogenase in the active form (PDHA) was found to increase with [Ca2+] m, with a K0.5 for activation of approximately 300 nM-Ca2+. This value of the activation constant was not affected by conditions, e.g. addition of Mg2+, which changed the [Ca2+] m/[Ca2+] o concentration gradient, and the presence of different oxidizable substrates, which changed the [NADH/NAD+] m concentration ratio. Thus pyruvate dehydrogenase interconversion responds directly to changes in [Ca2+] m, as inferred in earlier work. |
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