THE HETEROGENEITY OF RAT BRAIN MITOCHONDRIA ISOLATED ON CONTINUOUS SUCROSE GRADIENTS¹

Abstract

Abstract— The distribution of a series of enzymes in the post‐nuclear supernatant of rat brain homogenates was investigated following continuous density‐gradient centrifugation. The enzymes studied were acetyl coenzyme A synthetase, glutamic dehydrogenase, glutamine synthetase, glutaminase I, succinic dehydrogenase and monoamine oxidase. Each of these enzymes with the exception of glutamine synthetase appears predominantly in the mitochondrial region of the gradient. Although about 20 per cent of this enzyme is present in the crude mitochondrial pellet, on density gradient centrifugation no special association of glutamine synthetase with any of the mitochondrial fractions was observed. Each of the other enzymes studied was found to have a characteristic distribution in the gradient; this suggests that brain mitochondria may be heterogeneous both in buoyant density and in their enzyme content. Three principal fractions are described: (i) dense particles containing high concentrations of acetyl coenzyme A synthetase and glutamic dehydrogenase; (ii) a fraction comprising the bulk of the mitochondria with high levels of monoamine oxidase, succinic dehydrogenase and glutaminase I; and (iii) particles in the synaptic ending region of the gradient characterized by relatively high levels of monoamine oxidase and succinic dehydrogenase and containing only small amounts of the other enzymes studied. If the mitochondrial heterogeneity that is observed on centrifugation reflects the existence within brain cells of mitochondria with specialized function, a partial explanation may be available for multiple pools of tricarboxylic acid cycle intermediates which have been postulated from isotopie labelling experiments.

Heterogeneity of mitochondria in rat brain

“…Previously it has been established [2] that acetylCoA synthetase is distributed in a way similar to GDH. From these results and a study of the incorporation of specifically labelled glucose and acetate into glutamate, glutamine and asparate in mice brain [5,6], it was concluded that at least two distinct tricarboxylic acid cycles were present in brain.…”

“…All enzymes, except MAO and glutaminase, were incubated with 0.5% Triton-X-100 at 4 ° for 30 min prior to assay. 2 In sucrose density gradient centrifugations in swing out rotors Fonnum [21] found for the mitochondrial enzyme fumarase two peaks, Van Kempen et al [8] found separated peaks for two different mitochondrial enzymes, cytochrome oxidase and 7-aminobutyric acid transaminase (GABAT). The distribution curves can be described as Gaussian curves: two for the "free" mitochondria and the third one for the synaptosomal mitochondria.…”

“…Evidence has been presented by Salganicoff and De Robertis [1] and Neidle et al [2] that in sucrose density gradients of brain mitochondrial fractions glutamate dehydrogenase (GDH) and succinate dehydrogenase (SDH) are not distributed in the same way. With zonal sucrose density gradient centrifugation liver mitochondria have been shown to be heterogeneous (Swick et al [3]).…”

“…We therefore decided to apply the same technqiue to rat brain preparations. From results given by Salganicoff and Koeppe [4] and Neidle et al [2], it is possible to deduce that isocitrate dehydogenase-NADP (ICD-NADP) and GDH are localized in a population of mitochondria, differing from that containing the major amount of SDH, isocitrate dehydrogenase-NAD (ICD-NAD) and monoamine oxidase (MAO).…”

Resolution of Three Distinct Populations of Nerve Endings From Rat Brain Homogenates by Zonal Isopycnic Centrifugation

“…These would then represent a special kind of mitochondrion biochemically distinguishable from free brain mitochondria . There is indeed evidence in the literature suggesting that intraterminal mitochondria strongly differ in their enzymatic compcsition from perikarial mitochondria (34,35) . Finally, there remains the possibility that in adrenergic nerve endings, MAO may be partially localized in structures other than mitochondria ; for instance, associated with the nerve ending membranes or storage vesicles .…”