Murray Strassman

Accumulation of α-ketoglutaric acid in yeast mutants requiring lysine☆

Accumulation of a-ketoglutaric acid in two nonallelic yeast mutants (Ly7 and Ly4) requiring lysine has been demonstrated. In addition to α-ketoglutarate, Ly7 accumulates homocitrate, and Ly4 accumulates both homocitrate and cis-homoaconitate. The cell-free preparations of Ly4 lack homoacunitase activity, but Ly7 has homocitrate synthetase and homoaconitase activities. The mutants that fail to accumulate homoisocitrate have so far been found to accumulate α-ketoglutarate, which is not accumulated in wild-type culture. The accumulation of α-ketoglutarate has been confirmed in radioactive and nonradioactive experiments, and the mutant product has been identified by various Chromatographic and chemical procedures. The accumulation data for these mutants as well as for Ly12 suggest that there are two different enzymic steps to convert homocitrate to homisocitrate. The accumulation of α-ketoglutarate has been found in mutants which are blocked at steps preceding the synthesis of homoisocitrate.

A study of acetyl-CoA condensation with α-keto acids

Abstract The condensation of acetyl-CoA with α-ketobutyrate and with α-ketovalerate to yield α-ethylmalic acid and α-( n -propyl)malic acid, respectively, has been demonstrated to occur in the presence of dialyzed cell-free extracts of bakers yeast ( Sacchararomyces cerevisiae ). These reactions have been studied by means of a fluorometric assay procedure suitable for determination of α substituted malic acid derivatives. The reactions were shown to require α-keto acid, acetate, CoA, and ATP or the presence of α-keto acid and acetyl-CoA alone. The reaction between acetyl-CoA and α-ketobutyrate was also demonstrated by means of acetyl-CoA disappearance as measured by a decrease in absorbance at 232 mμ. Comparison of specific activities of the various ammonium sulfate fractions of the yeast extract indicated that the condensations of acetyl-CoA with α-ketobutyrate and α-ketovalerate are catalyzed by the same enzyme fraction. In the light of these results and results of experiments reported previously the condensation of acetyl-CoA with α-keto acids may be viewed as a general type of enzyme reaction.

Fluorometric assay of malic acid and substituted derivatives

Abstract A fluorometric assay is described for the determination of α- and β-substituted malic acids. The procedure involves production of a β-keto acid and subsequent condensation with resorcinol to yield fluorescent derivatives of 7-hydroxycoumarin. The assay was shown to be useful over a wide range of concentrations, and is sensitive enough to be used for less than microgram quantities of α-substituted malic acids.

[70] Fluorometric assay of malic acid and its α-substituted derivatives

Publisher Summary The chapter discusses principle and procedure for fluorometric assay of malic acid, and its α-substituted derivatives. This fluorometric assay is based on the degradation of substituted malic acids to β-keto acids, which condenses with resorcinol to form derivatives of the highly fuorescent 7-hydroxycoumarin. This procedure is not applicable to α-substituted malic acids as they are degraded extensively in hot acid. The use of cold concentrated sulfuric acid limits the degradation of α-substituted malic acids to β-keto acids. Acetoacetic acid, the simplest β-keto acid, condenses with resorcinol in the presence of acid at room temperature to form 7-hydroxycoumarin. α-Substituted malic acids such as α-ethyl malic, α-methyl malic, α-isopropylmalic, and citric acids show a much higher degree of fluorescence than β-substituted malic acids such as β-methyl malic and β-ethyl malic acid. Malic acid shows a still lower degree of fluorescence. The relationship of fluorescence to concentration is linear for all these compounds.