The Metabolism of Rat Brain Mitochondria

TLDR

Previous studies used crude brain mitochondrial preparations; this work develops a rapid Ficoll gradient method to isolate purified rat cortical mitochondria and notes that increasing K⁺ in the medium enhances oxygen uptake. Mitochondria were isolated by a rapid Ficoll gradient and characterized for nucleotide levels, cytochrome composition, and K⁺‑dependent respiration using pyruvate plus malate. The purified mitochondria were free of contamination, exhibited high respiratory control ratios (>7) with pyruvate plus malate, had cytochrome ratios comparable to other mammals, and K⁺ increased respiration by enhancing substrate availability rather than electron transport.

Abstract

Abstract Since most previous studies on brain mitochondria have used a relatively crude preparation, a method was developed for preparing a purified mitochondrial fraction from rat brain cerebral cortex utilizing a rapid simple discontinuous Ficoll density gradient procedure. This mitochondrial preparation was shown by electron microscopy and enzymatic assay to be (a) free from contamination by synaptosomes or other membranous fragments and (b) predominantly of neuroglial origin. These mitochondria exhibited good respiratory control and respiratory rates with various substrates (pyruvate g succinate g glutamate g citrate, acetylcarnitine, isocitrate, α-ketoglutarate, α-glycerophosphate), comparable with other mammalian mitochondria. Pyruvate plus malate was oxidized at rates higher than any of the other substrates tested and consistently showed respiratory control ratios greater than seven. The mitochondria were further characterized with respect to nicotinamide and adenine nucleotide content, and cytochrome complement and content. The ratios of cytochrome b:c1:c:a:a3 were 0.6:0.9:1.7:1:0.6, similar to those observed in other mammalian mitochondria. As has been noted previously with brain preparations, increasing the K+ in the incubation medium caused an increase in the oxygen uptake with all the above substrates (except α-glycerophosphate). This effect was studied in more detail with pyruvate plus malate as substrate. Half-maximal respiration rates were obtained with 10 to 20 mm K+ in both ADP and valinomycin-stimulated mitochondria. The effect of K+ was not directly associated with an activation of electron transport per se, but rather with an increase in the availability of substrate for oxidation.