Brain α‐Ketoglutarate Dehydrogenase Complex: Kinetic Properties, Regional Distribution, and Effects of Inhibitors

Abstract

The substrate and cofactor requirements and some kinetic properties of the alpha-ketoglutarate dehydrogenase complex (KGDHC; EC 1.2.4.2, EC 2.3.1.61, and EC 1.6.4.3) in purified rat brain mitochondria were studied. Brain mitochondrial KGDHC showed absolute requirement for alpha-ketoglutarate, CoA and NAD, and only partial requirement for added thiamine pyrophosphate, but no requirement for Mg2+ under the assay conditions employed in this study. The pH optimum was between 7.2 and 7.4, but, at pH values below 7.0 or above 7.8, KGDHC activity decreased markedly. KGDHC activity in various brain regions followed the rank order: cerebral cortex greater than cerebellum greater than or equal to midbrain greater than striatum = hippocampus greater than hypothalamus greater than pons and medulla greater than olfactory bulb. Significant inhibition of brain mitochondrial KGDHC was noted at pathological concentrations of ammonia (0.2-2 mM). However, the purified bovine heart KGDHC and KGDHC activity in isolated rat heart mitochondria were much less sensitive to inhibition. At 5 mM both beta-methylene-D,L-aspartate and D,L-vinylglycine (inhibitors of cerebral glucose oxidation) inhibited the purified heart but not the brain mitochondrial enzyme complex. At approximately 10 microM, calcium slightly stimulated (by 10-15%) the brain mitochondrial KGDHC. At concentrations above 100 microM, calcium (IC50 = 1 mM) inhibited both brain mitochondrial and purified heart KGDHC. The present results suggest that some of the kinetic properties of the rat brain mitochondrial KGDHC differ from those of the purified bovine heart and rat heart mitochondrial enzyme complexes. They also suggest that the inhibition of KGDHC by ammonia and the consequent effect on the citric acid cycle fluxes may be of pathophysiological and/or pathogenetic importance in hyperammonemia and in diseases (e.g., hepatic encephalopathy, inborn errors of urea metabolism, Reye's syndrome) where hyperammonemia is a consistent feature. Brain accumulation of calcium occurs in a number of pathological conditions. Therefore, it is possible that such a calcium accumulation may have a deleterious effect on KGDHC activity.

Selective Reductions in the Activity of the Pyruvate Dehydrogenase Complex in Mitochondria Isolated from Brain Subregions following Forebrain Ischemia in Rats

“…Consistent with previous investigations (Sims, 1990), the specific activities of mitochondrial en zymes measured in the present study were two-to threefold greater than values reported previously (Lai and Cooper, 1986;Mizuno et aI., 1987;Elnageh and Gaitonde, 1988) for brain mitochondria isolated using alternative preparative procedures. As the isolated mitochondrial population is likely to in clude organelles from both neuronal and glial cells, no conclusions can be reached as to the cellular location of the observed changes in the dorsolateral striatum.…”

“…The activity of a-ketoglutarate dehydrogenase in mito chondria was determined spectrophotometric ally as de scribed by Lai and Cooper (1986). Only one cycle of freezing and thawing was required to obtain maximum activity.…”

Keeping cool in acute liver failure: Rationale for the use of mild hypothermia

“…Increased brain lactate correlated with worsening encephalopathy and intracranial hypertension in experimental models [92] and [121], and it was due to increased de novo synthesis from circulating glucose. This observation suggests decreased oxidation of pyruvate [92], in accordance with inhibition of alpha-ketoglutarate dehydrogenase and stimulation of the glycolytic enzyme phosphofructokinase by ammonia in vitro [122] and [123]. The effects of hypothermia (32-33 °C) on brain glucose metabolism were studied by Jalan et al in patients with ALF and uncontrolled ICP [80].…”

Hypothermia in Acute Liver Failure

“…It has also been demonstrated that alanine is elevated in the hyperammonemic brain (Hilgier et al, 1999;Mans et al, 1994;Swain et al, 1992) and more recently in rats with ALF using nuclear magnetic resonance (NMR) spectroscopy (Chatauret et al, 2003). Increased lactate and alanine production indirectly suggest that anaerobic pathways (increased glycolytic activity) may be stimulated to compensate for a decreased pyruvate oxidation (due to ammonia inhibition on the enzyme alpha-ketoglutarate dehydrogenase in the tricarboxylic acid cycle (Lai and Cooper, 1986)) and maintain ATP production. Furthermore, decreased concentrations of the neuronal marker molecule N-acetylaspartate (NAA) have been demonstrated in frontal cortex of rats in coma stages of HE as a result of ALF, reflecting neuronal mitochondrial dysfunction (Chatauret et al, 2003).…”