Subject(s)
Brain/enzymology , Coenzyme A Ligases/analysis , Acetate-CoA Ligase/analysis , Acetates , Animals , Carbon Radioisotopes , Chromatography, Ion Exchange , Kinetics , Methods , RatsABSTRACT
Acetyl-CoA synthase (EC 6.2.1.1), Propionyl-CoA synthase (EC 6.2.1.-) and butyryl-CoA synthase (EC 6.2.1.2) were measured in subcellular fractions prepared by primary and density-gradient fractionation from adult rat brain by a method resulting in recoveries close to 100%. Most of the activity of the three enzymes was recovered in the crude mitochondrial fraction. On subfractionation of this crude mitochondrial fraction with continuous sucrose density gradients, most of the activity of the three enzymes was found at a higher density than NAD+-isocitrate dehydrogenase and at about the same density as glutamate dehydrogenase, confirming earlier reported data for acetyl-CoA synthase. The finding that propionyl-CoA synthase and butyryl-CoA synthase had about the same distribution in the gradients as acetyl-CoA synthase adds support to the hypothesis that mitochondria involved in the metabolism of these short-chain fatty acids (all three of which have been shown to result in a rapid and high labelling of glutamine in vivo) form a distinct subpopulation of the total mitochondrial population. The three synthase activities were found to differ from each other in their rate of change and their subcellular localization during rat brain development. This, in combination with the observation that in gradients of adult brain preparations the three activities did not completely overlap, suggests that the three synthase activities are not present in the same proportion to each other in the same subpopulation (s) of mitochondria in the brain.
Subject(s)
Acetate-CoA Ligase/metabolism , Brain/enzymology , Coenzyme A Ligases/metabolism , Aging , Animals , Brain/growth & development , Cell Nucleus/enzymology , Centrifugation, Density Gradient , Female , Isocitrate Dehydrogenase/metabolism , L-Lactate Dehydrogenase/metabolism , Male , Microsomes/enzymology , Mitochondria/enzymology , Proteins/analysis , Rats , Time FactorsABSTRACT
The subcellular localizations of gamma-aminobutyrate transaminase (EC 2.6.1.19) and glutamate dehydrogenase (EC 1.4.1.2) in brain tissue of adult rats were compared with each other and with those of NAD+-isocitrate dehydrogenase (EC 1.1.41) and monoamine oxidase (EC 1.4.3.4; kynuramine as substrate). Crude mitochondrial fractions from brain tissue were centrifuged in continuous sucrose density gradients. gamma-Aminobutyrate transaminase and glutamate dehydrogenase were always found at a higher density than NAD+-isocitrate dehydrogenase and monoamine oxidase. When centrifuged for 1 h at 53 000gav., there was a slight difference between the distribution profiles of glutamate dehydrogenase and gamma-aminobutyrate transaminase. This difference was larger when the centrifugation time was only 15 min. It is concluded that there are subpopulations of brain mitochondria with differing proportions of gamma-aminobutyrate transaminase and glutamate dehydrogenase. The results are discussed in relation to evidence obtained with labelled precursors in vivo that there are at least two small glutamate compartments in adult brain.
