Louis N. Ceci

Homocitrate synthase from yeast.

Homocitrate synthase activity has been partially purified from yeast. Homocitrate synthase activity was sensitive to inhibition by lysine both in vivo and in vitro only at high concentrations of lysine. Two enzyme activities have been demonstrated in several strains of yeast by isoelectric focusing. The two enzymes were equally sensitive to inhibition by lysine. One enzyme activity was repressed to a greater extent when the culture medium was supplemented with excess lysine.

Control of lysine biosynthesis in yeast

Abstract The inhibition of the pathway of biosynthesis of lysine in yeast has been studied in vivo . The site sensitive to inhibition at low concentrations of lysine is after α-aminoadipate on the pathway. A test was made for a branch point in the pathway, but no evidence was obtained to demonstrate a branch point.

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.

[78] Preparation of homocitric, homoaconitic, and homoisocitric acids

Publisher Summary The chapter discusses the method for preparation of homocitric, homoaconitic, and homoisocitric acids. Homoisocitric acid is obtained by reduction of triethyl 2-oxaloglutarate, prepared by condensation of diethyl glutarate with diethyl oxalate in the presence of sodium ethoxide. The free acid is obtained from the triester of homoisocitrie acid by hydrolysis. Homoaconitic acids are prepared by dehydrating triethyl homoisoeitrate with acetyl chloride, followed by hydrolysis to the free acids. The cis and trans isomers of homoaconitic acid are separated by anion-exchange chromatography; trans-homo-aconitic acid is eluted first from a column of Dowex 1-formate. Homocitric acid is prepared by treating diethyl B-ketoadipate with hydrogen cyanide, followed by hydrolysis of the cyanohydrin to the free acid. The intermediate diethyl B-ketoadipate is prepared by condensing magnesium malonic ester with p-carbethoxypropionyl chloride, followed by thermal decomposition of diethyl p-keto-a-carbethoxyadipate in the presence of β-naphthalenesulfonic acid.

[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.