ABSTRACT
The products of the reactions of mitochondrial 2-oxo acids with hydrogen peroxide and tert-butyl hydroperoxide (tert-BuOOH) were studied in a chemical system and in rat liver mitochondria. It was found by HPLC that the decarboxylation of alpha-ketoglutarate (KGL), pyruvate (PYR), and oxaloacetate (OA) by both oxidants results in the formation of succinate, acetate, and malonate, respectively. The two latter products do not metabolize in rat liver mitochondria, whereas succinate is actively oxidized, and its nonenzymatic formation from KGL may shunt the tricarboxylic acid (TCA) cycle upon inactivation of alpha-ketoglutarate dehydrogenase (KGDH) under oxidative stress, which is inherent in many diseases and aging. The occurrence of nonenzymatic oxidation of KGL in mitochondria was established by an increase in the CO(2) and succinate levels in the presence of the oxidants and inhibitors of enzymatic oxidation. H(2)O(2) and menadione as an inductor of reactive oxygen species (ROS) caused the formation of CO(2) in the presence of sodium azide and the production of succinate, fumarate, and malate in the presence of rotenone. These substrates were also formed from KGL when mitochondria were incubated with tert-BuOOH at concentrations that completely inhibit KGDH. The nonenzymatic oxidation of KGL can support the TCA cycle under oxidative stress, provided that KGL is supplied via transamination. This is supported by the finding that the strong oxidant such as tert-BuOOH did not impair respiration and its sensitivity to the transaminase inhibitor aminooxyacetate when glutamate and malate were used as substrates. The appearance of two products, KGL and fumarate, also favors the involvement of transamination. Thus, upon oxidative stress, nonenzymatic decarboxylation of KGL and transamination switch the TCA cycle to the formation and oxidation of succinate.
Subject(s)
Mitochondria, Liver/metabolism , Oxidative Stress/physiology , Succinic Acid/metabolism , Amination , Animals , Citric Acid Cycle/drug effects , Decarboxylation , Glutamic Acid/metabolism , Hydrogen Peroxide/metabolism , Hydrogen Peroxide/pharmacology , Ketoglutarate Dehydrogenase Complex/antagonists & inhibitors , Ketoglutarate Dehydrogenase Complex/metabolism , Ketoglutaric Acids/metabolism , Liver/enzymology , Male , Oxaloacetic Acid/metabolism , Oxidants/metabolism , Oxidants/pharmacology , Pyruvic Acid/metabolism , Rats , Rats, Wistar , Reactive Oxygen Species/metabolism , tert-Butylhydroperoxide/metabolism , tert-Butylhydroperoxide/pharmacologyABSTRACT
BACKGROUND: It is generally accepted that the glyoxylate cycle exists in microorganisms and higher plants but absent in higher animals. the hypodhesis of the glyoxylate cycle in the tissues of higher animals with a high level of physiological activity was first proposed by Kondrashova and Rodionova in 1971. The goal of this work was yo verifv this in newborn rats, which possess a 2.5-fold hygher physiological activity and oxygen consumption rate than adult rats. MATERIAL/METHODS: Newborn (7-day-old) anradult 1 ats were used for this experiment. The activities of the key enzymes of the glyoxylate cocle (isecitrate lyse and nmalate synthase) were measured by HPLC and spectroscopic methods. The activities of isocitrate lyase and malate synthase were found in the liver homogenates prepared from newborn rats, but not from adult rats. The activities of the enzymes common to both the Krebs cycle and the glyoxylate cycle (citrate synthase, aconitase, and malate dehydrogenase) were 20-40% higher in newborn than in adult rats. CONCLUSIONS: These data indicate the existence of the glyoxylate cycle in animal tissues.
