Abstract
The ratio of free ATP to free ADP in the mitochondrial matrix [( ATPf]/[ADPf]) has been measured in suspensions of isolated mitochondria under conditions of active phosphorylation of extramitochondrial ADP. These measurements utilized phosphoenolpyruvate carboxykinase which is present in the matrix of mitochondria from the livers of guinea pigs, chickens, and pigeons. Mitochondria isolated from these sources also contain nucleoside diphosphate kinase, malate dehydrogenase, and glutamate dehydrogenase or 3-OH-butyrate dehydrogenase. Together these enzymes catalyze the synthesis of phosphoenolpyruvate and CO2 from oxaloacetate with oxidative phosphorylation as an energy source. These reactions have been shown to be fully reversible in suspensions of mitochondria isolated from the above sources. With oxidative phosphorylation as the source of ATP, phosphoenolpyruvate was synthesized from malate and conversely addition of phosphoenolpyruvate, ADP, and CO2 led to synthesis of malate and ATP. The forward and reverse reactions were allowed to continue until the rate of change of metabolite concentrations was minimal and then the latter were measured. The intramitochondrial [Mg-ATPf]/[MgADPf] was calculated from the equilibrium constants for the reactions and the measured steady state concentrations of the metabolites in both the intra- and extramitochondrial spaces. The value of the intramitochondrial [MgATPf]/[MgADPf] was found to exceed the extramitochondrial value (adjusted to the same free Mg2+ concentration) by a factor (+/- S.E.) of 0.83 +/- 0.22 (n = 17) for the forward reaction and 1.81 +/- 0.54 (n = 11) for the reverse reaction. It is concluded that the adenine nucleotide translocase catalyzes electroneutral exchange of ATP for ADP and that this reaction does not contribute significantly to the energetics of mitochondrial oxidative phosphorylation.
Highlights
The ratioof free ATP to freeADP in the mitochon- of ATP, ADP, and orthophosphate (Pi) across the inner drialmatrix ([ATPt]/[ADP,]) has been rfieasured in mitochondrial membrane
We report measurements of intramitochondrial [ATPf]/[ADPf] using the combination of two
We show thatthe intramitochondrial [ATPf]/[ADPf] is much higher than thatreported for the totalnucleotides and is equal to or slightly greater than thecomparable extramitochondrial [ATP]/[ADP] under conditions of active phosphorylation of extramitochondrial ADP
Summary
Methods tralized to pH 6.5-7.0 with a 3 M KzC03-0.5M triethanolamine base mixture, the KClO, precipitate was removed by centrifugation, and Determination of K4 for P-enolpyruvate Carboxykime-P-enol- the metabolite assays were carried out on the neutralized samples. Mitochondrial Membrane Potential and pH Gradients-At the end of the incubations and assamples were being quenched for metabolite assays, 1-ml fractions were treated for 60 s with 2.5 pM (TPMP+). Dehydrogenase, 3-OH-butyrate Dehydrogenase, and Nucleoside Diphosphate Kinase-The equilibrium constant for the glutamate dehydrogenase reaction (Equation 6) at 27 "C has been reported by. Malate dehydrogenase reaction (Equation 5) as measured by Guynn et af.[46] is 1.02 X 10"* M a t 25 and 2.9 X 10"' M at 38 Values of1.52 X 10"' M and 2.28 X 10"' M for 30 and 35 "C, respectively, have been used for the calculations in this paper. The equilibrium constant for the 3-OH-butyrate dehydrogenase reaction (Equation 7). All other chemicals were of the highest quality available commercially
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