THE NON-ENZYMATIC OXIDATION OF a-KETO- GLUTARATE* I. THE EFFECTS OF MANGANOUS IONS AND AMINO ACIDS BY GEORGE KALNITSKY

With a washed and dialyzed preparation of rabbit kidney cortex mitochondria, the requirement of Mg ++ (or R/In++), inorganic phosphate, and adenosinetriphosphate for the enzymatic oxidation of a-ketoglutarate to succinate plus COZ may be readily demonstrated (2), confirming results (3) with cat heart muscle. Diphosphothiamine is a necessary coenzyme for this enzymatic system (4, 5). However, in control vessels with no enzyme present, an appreciable oxygen uptake was recorded (2). This interesting observation stimulated investigation of the problem, the results of which are reported here. E$ecl of M&+-In the presence of a-ketoglutarate, MnC12 or MnS04, and Verona1 buffer, with no tissue or enzyme present, oxygen is taken up, as measured in the conventional Warburg apparatus (Table I). When either a-ketoglutarate or Mn++ was omitted or allowed to remain in the side arm of the Warburg flask, there was no oxygen taken up. Therefore, both a-ketoglutarate and Mn++ are necessary for this non-enzymatic oxidation. The rate of oxidation is increased by increasing the concentration of a-ketoglutarate or of R/In++ (Table I). Mg++, at various concentrations, will not replace Mn++ (Table I). Unless otherwise noted, all the experiments were carried out at 30.1°, in an atmosphere of air, and a total volume of 2.0 ml. with 0.3 ml. of KOH in the alkali well. IdentiJication of a-KetogZuturate-Several different samples of a-ketoglutarate obtained from the Nutritional Biochemicals Corporation and synthesized in our laboratory, and an authentic sample kindly supplied by Dr. Van R. Potter, all behaved similarly. The a-ketoglutarate was recrystallized from acetone and benzene (6) and melted at 112-113’. The melting point reported is 112” (6). The melting points of mixtures of several different samples showed no depression. The melting point of the 2,4-dinitrophenylhydrazone was 215” (reported, 218” (7)). The free acid had the correct neutralization equivalent and the absorption spectrum of its 2,4-dinitrophenylhydrazone was identical with that reported (8, 9).