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Abstract
Abstract Evidence is presented in support of the hypothesis that the pyruvate dehydrogenase multienzyme complex of intact rabbit heart mitochondria may be regulated by a phosphorylation-dephosphorylation mechanism. Mitochondria incubated in the presence of pyruvate plus l-malate and either ADP or uncoupler exhibit nearly identical, rapid rates of pyruvate oxidation but possess markedly different ATP levels. It was shown that under metabolic conditions which lead to a release or mobilization of intramitochondrial magnesium and which also have a high intramitochondrial ATP level, pyruvate oxidation was nearly completely inhibited after a brief lag phase. It was shown that the addition of exogenous magnesium to uncoupled mitochondria supplemented with ADP caused a more rapid inhibition of pyruvate oxidation. The observed inhibition of pyruvate oxidation was dependent upon the time of preliminary incubation with uncoupler and ADP and was atractyloside-sensitive. Evidence was obtained indicating that the inhibition of pyruvate oxidation was specific for the substrate, pyruvate, i.e. the oxidation of l(-)palmitylcarnitine or α-ketoglutarate was unaffected under conditions leading to an inhibition of pyruvate oxidation. In addition, it was demonstrated that the inhibition of pyruvate oxidation was not caused by an accumulation of either NADH or acetyl-CoA, both inhibitory products of the pyruvate dehydrogenase reaction, during the course of these experiments. The experiments reported in this communication indicate that the control of the availability of both ATP and magnesium are crucial for the regulation of the pyruvate dehydrogenase multienzyme complex. These studies are consistent with the possibility that the pyruvate dehydrogenase-linked protein kinase may be an effective means of regulating the conversion of pyruvate to acetyl-CoA in intact metabolic systems such as the isolated mitochondrion.
Highlights
MethodsRabbit heart mjtochondria were prepared according to the procedure of Van Korff [13] using a homogenization medium containing KCl, 0.18 M; EDTA, 5 InM; and defatted [14] bovine serum albumin, Fraction V, 0.5’;); w/v
ADP or uncoupler exhibit nearly identical, rapid rates of pyruvate oxidation but possess markedly different ATP
It was shown that the addition of exogenous magnesium to uncoupled mitochondria supplemented with ADP caused a more rapid inhibition of pyruvate oxidation
Summary
Rabbit heart mjtochondria were prepared according to the procedure of Van Korff [13] using a homogenization medium containing KCl, 0.18 M; EDTA, 5 InM; and defatted [14] bovine serum albumin, Fraction V, 0.5’;); w/v. Isolated mitochondria were used for the experiments within 2 hours after their isolation and were stored without aeration at a mitochondrial protein concentration of from 15 to 30 mg per ml prior to their use in the experiments. Prot,ein concentrations were estimated using a biuret procedure [15]. Samples (2.5 ml) of the mitochondrial incubations mere n-ithdrawn rapidly and were deproteinized with perchloric acid (6% w/v). The perchloric acid extracts were neutralized with a solution containing 3 ix potassium carbonate plus 0.5 BI tri-
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