flavin-adenine-dinucleotide and 1-10-phenanthroline

Benziman, Moshe (The Hebrew University of Jerusalem, Jerusalem, Israel), and Y. Galanter. Flavine adenine dinucleotide-linked malic dehydrogenase from Acetobacter xylinum. J. Bacteriol. 88:1010-1018. 1964.-The properties of the pyridine nucleotide-nonlinked malic dehydrogenase of Acetobacter xylinum were investigated in the supernatant fluid obtained by high-speed centrifugation of sonic extracts. Ferricyanide, phenazine methosulfate, and to a lesser extent dichlorophenolindophenol were active as oxidants for malate oxidation. After acid ammonium sulfate precipitation, the enzyme lost its malate-oxidizing activity. The enzyme was reactivated by low concentrations of flavine adenine dinucleotide (FAD) but not by flavine mononucleotide (FMN) or riboflavine. Atabrine inhibited the enzyme, and the inhibition was relieved by FAD but not by FMN or riboflavine. Malate-oxidizing activity was inhibited by hematin. The inhibition was prevented by imidazole or globin. o-Phenanthroline, 8-hydroxy quinoline, alpha,alpha'-dipyridyl, and p-chloromercuribenzoate inhibited malate oxidation. Amytal markedly inhibited oxidation of malate in the presence of oxygen, phenazine methosulfate, or dichlorophenolindophenol, but not in the presence of ferricyanide. The results suggest that the malic dehydrogenase of A. xylinum is a FAD enzyme, which contains an ironbinding site essential for its activity. Nonheme iron and sulfhydro groups are possibly involved in enzyme activity. The malic dehydrogenase is functionally linked to the cytochrome chain.

Topics: Acetobacter; Adenine; Amobarbital; Enzyme Inhibitors; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Gluconacetobacter xylinus; Heme; Imidazoles; Iron; Israel; Malate Dehydrogenase; Malates; Pharmacology; Phenanthrolines; Phenazines; Phenols; Quinacrine; Quinolines; Research; Riboflavin

Furuya, Akira (University of Illinois College of Medicine, Chicago) and James A. Hayashi. Glycolic acid oxidation by Escherichia coli adapted to glycolate. J. Bacteriol. 85:1124-1131. 1963.-A procedure is described for extraction and partial purification of glycolic acid oxidase from Escherichia coli adapted to grow on glycolate as the sole carbon source. Enzyme activity was assayed by oxygen uptake and by reduction of 2,6-dichlorophenol-indophenol. Glyoxylic acid was the product of glycolate oxidation by the enzyme. Enzyme activity, which diminishes rapidly on storage, shows a maximum at pH 6 to 7. We were unable to show any cofactor requirement. Compounds which inhibited glycolate oxidation and their order of inhibitory activity were: p-hydroxymercuribenzoate > sodium azide > iodoacetate and o-phenanthroline > ethylenediaminetetraacetic acid. Tests of enzyme specificity showed that the following compounds were oxidized, but at different rates: glycolate, d-lactate, l-lactate, dl-alpha-hydroxybutyrate, dl-malate, and dl-glycerate. Citrate, tartrate, and dl-beta-hydroxybutyrate were not oxidized. Potassium cyanide stimulated oxygen uptake when glycolate and lactate were oxidized. Whether the oxidations were due to different oxidases or to a single oxidase with a wide range of specificities was tested by observing the oxidation of glycolate, d-lactate, and l-lactate under various conditions. Ammonium sulfate fractionation of a crude extract did not change the relative ability to oxidize the three acids. However, the three oxidative capacities diminished at different rates during storage at 0 C for 6 days. The partially purified glycolic oxidase preparations were probably mixtures of several different oxidases.

Topics: Azides; Citrates; Cyanides; Edetic Acid; Enzyme Inhibitors; Escherichia coli; Flavin Mononucleotide; Flavin-Adenine Dinucleotide; Glycolates; Hydroxybutyrates; Indophenol; Iodoacetates; Lactates; Malates; Manometry; NAD; NADP; Oxidation-Reduction; Oxidoreductases; Phenanthrolines; Potassium; Research; Tartrates