In vitro 14C-leucine incorporation into nonsynaptic and synaptic rat brain mitochondria isolated by Ficoll gradients

Abstract

The in vitro incorporation of 14C-leucine by nonsynaptic and synaptic rat brain mitochondria purified by means of discontinuous Ficoll gradients has been characterised.The incorporation was linear for the first 45 min for both populations.Synaptic mitochondria showed a higher rate of incorporation than the nonsynaptic mitochondria at high concentrations of leucine.The incorporation was more effective in the presence of Mg2+ and inhibited by dinitrophenol.The incorporation was sensitive to chloramphenicol and insensitive to cycloheximide.Bacterial contamination was in any case lower than 1,000 colonies per ml after the incubation period.The incorporation was carried out in the presence of either an external ATP-generating system consisting of ATP, phosphoenolpyruvate and pyruvate kinase or with mitochondria respiring with oxidisable substrates plus ADP (state III).The rates obtained for incorporation in this state III were higher for all the substrates assayed (succinate, pyruvate and glutamate) than in the presence of exogenous ATP.The highest rate obtained was found when glutamate was the respiratory substrate.No significant metabolic oxidation of leucine occurs in either synaptic or nonsynaptic mitochondria in the presence of exogenous ATP.Glutamate did not increase leucine uptake in any mitochondrial populations.