Estimation of l-malate and fumarate by malic decarboxylase of Lactobacillus arabinosus

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Abstract
The decarboxylase method for determining the sum of L-malate and fumarate was modified so that L-malate and fumarate could be measured separately. Malic acid was measured manometrically using washed suspensions of malate-adapted cells of L. arabinosus 17-5 at 35[degree] in a soln. at pH 5.0 containing 2.5 x 10-3 [image] glucose, 2.5 x 10-3 [image] manganous chloride and 10-3 [image]. KCl. Max. yields of the enzyme were obtained by growing the organism for 24 hrs. at 30[degree] on media containing 10-1 [image] dL-malate, with a 2d addition of 0.1 vol. [image] DL-malate, 12 hrs. after inoculation. Oxaloacetate, pyruvate and fumarate also formed CO2 Oxaloacetate could be removed with aniline, pyruvate fixed with hydroxylamine or semicarbazide and fumarate removed by reduction with Zn and phosphoric acid, so that malate could be measured accurately in samples containing these acids. Washed cells retained most of their malic decarboxylase activity for several weeks at 2[degree] when suspended in 0.1 [image] KCl. Inactive cells could be partly reactivated by addition of cozymase or incubation with glucose and nicotinic acid. Of some 40 substances tested only n-hexadecyltrimethyl-ammonium bromide (cetavlon), iodoacetate, tungstate, trichloro-acetate, trioctylamine, 2,4-dinitrophenol, CuSO4, aluminum sulfate and FeSO4 inhibited sufficiently at 10-2 [image] to interfere with the estimation; at 10-4 [image] only cetavlon inhibited severely. The sum of fumarate and malate was measured by adding an excess of fumarase, and the fumarate content is obtained by difference. Fumarase was progressively inactivated by suspensions of L. arabinosus at pH 5.0 and 30[degree]. Animal tissues were found to contain between 1 and 13 [mu] mol./g. wet weight malate plus fumarate. All plant materials tested contained malate (between 10 and 200 [mu] mol./g. wet weight), while fumarate was absent from most materials. The occurrence of fumarate in Myrrhis odorata and Glaucium flavum (13 and 99 u mol./g. wet weight respectively) was confirmed. The hemolymph of Gastrophilus intestinalis larvae contained exceptionally high concns. of malate and fumarate (24-38 [mu] mol./ml.); these acids account largely for the anion deficit reported by Levenbook (1950).