Concepedia

Regulation of Pyruvate Dehydrogenase in Rat Liver Mitochondria by Phosphorylation-Dephosphorylation

Elżbieta I. Wałajtys, Democleia P. Gottesman, John Williamson

Journal of Biological Chemistry · 1974 · 104 citations · 40 references

Open access

TL;DR

The study measured the proportion of active, non‑phosphorylated pyruvate dehydrogenase in intact rat liver mitochondria, quantifying ATP, ADP, Mg²⁺, and Ca²⁺ while incubating mitochondria with glutamate and malate under various metabolic states induced by oligomycin, uncouplers, ionophores, and inhibitors to manipulate intramitochondrial phosphorylation. Results show that pyruvate dehydrogenase activity is primarily controlled by kinase activity, with the active fraction increasing with ADP and decreasing with the ATP:ADP ratio, while phosphatase activity is only evident under severe Mg²⁺/Ca²⁺ depletion and normal Mg²⁺/Ca²⁺ levels maintain optimal phosphatase activity, allowing steady‑state activity to be governed by pyruvate and ADP inhibition of the kinase and avoiding unnecessary ATP consumption.

Abstract

Abstract The percentage of total pyruvate dehydrogenase in the active nonphosphorylated form was measured in intact rat liver mitochondria after rapid separation of the mitochondria through silicone oil and extraction with glycerol buffer at -10°. The contents of ATP, ADP, Mg2+, and Ca2+ in the mitochondrial matrix of separated mitochondria were determined in parallel experiments. Mitochondria were incubated with glutamate and malate as substrates in a variety of metabolic states. These were induced by addition of oligomycin, uncoupler, the divalent cation ionophore A23187, and other inhibitors in order to alter the phosphorylation state of the intramitochondrial adenine nucleotides and the contents of magnesium and calcium. Interconversion between the active nonphosphorylated form of pyruvate dehydrogenase and the inactive phosphorylated form appeared to be dominated by regulation of pyruvate dehydrogenase kinase activity. Under conditions of controlled respiration, the percentage of pyruvate dehydrogenase in the active form varied directly with the ADP content and inversely with the ATP:ADP ratio. Regulation of pyruvate dehydrogenase interconversion by pyruvate dehydrogenase phosphatase activity could only be demonstrated under severe conditions of magnesium and calcium depletion. It is concluded that the normal content of Mg2+ and Ca2+ in isolated mitochondria is sufficient to provide optimal activation of pyruvate dehydrogenase phosphatase and that release of Mg2+ by conversion of MgATP2- to ADP, Pi, and Mg2+ during active respiration has a negligible effect on pyruvate dehydrogenase phosphatase activity. The over-all steady state activity of pyruvate dehydrogenase appears to be determined by pyruvate and ADP which inhibit pyruvate dehydrogenase kinase, and thereby prevent phosphorylation and inactivation of pyruvate dehydrogenase. Regulation of pyruvate dehydrogenase activity is thus achieved without an unnecessary expenditure of ATP for enzyme phosphorylation and dephosphorylation.