Oxalate Decarboxylase Research Articles

New enzymic method for serum oxalate determination.

There have been several recent methods for determining serum oxalate in man (1-8), but reported normal values have ranged from 0.3 to 14 mg/liter. Recently Knowles and Hodgkinson (6) reported an enzymic method for oxalate determination in human serum by use of oxalate decarboxylase (EC 4.1.1.2) with subsequent colorimetric determination of the evolved CO2. The method is specific, but the technique is rather too elaborate for routine use. Our method also involves use of oxalate decarboxylase in a linked reaction with NAD+ requiring formate dehydrogenase (EC 1.2.1.2). Determinations are done spectrophotometrically, with reproducible results. We have recently applied the principle successfully for oxalate determinations in urine (9). In serum the mean value for data from 40 healthy volunteers was 1.82 mg/liter, the values for women being significantly higher than for men.

Enzymatic-potentiometric determination of oxalic acid

A method utilizing oxalate decarboxylase and potentiometry is described for the determination of oxalic acid. The oxalic acid was enzymatically decomposed to form formic acid and carbon dioxide. The released CO 2 was measured potentiometrically using a CO 2 electrode. A nernstian relationship between the voltage output of the CO 2 sensor and the concentration of oxalic acid, in the physiological range, 0.3–1.0 mg per 100 cm 3, has been observed.

Oxalate and glycolate synthesis by hemic cells.

Abstract Oxalate and glycolate were synthesized from 14 C-glyoxylate by leukocytes, erythrocytes, and dialyzed hemolysate preparations. Oxalate decarboxylase was used in a convenient and reliable assay of oxalate produced. Leukocytes were approximately 10 to 20 times as active in oxalate and glycolate synthesis, respectively, as erythrocytes. Data submitted to indicate that the erythrocytic enzyme system is lactic dehydrogenase (LDH) include nicotinamide, adenine dinucleotide dependence, pH curve, simultaneous glycolate and oxalate synthesis, inhibition by oxalate and oxamate, and coincidence of isoenzyme activities for lactate and glyoxylate. Coincidence of glyoxylate and lactate oxidation by LDH isoenzymes was also shown in leukocyte preparations. These findings are discussed in terms of the implications of the coupling by LDH of glyoxylate oxidation to oxalate and its reduction to glycolate or the competitive reduction of another substrate. The implications for the development of a pharmacologic inhibitor of oxalate synthesis are also presented.

Enzymatic oxalate decarboxylation in Aspergillus Niger: II. Hydrogen peroxide formation and other characteristics of the oxalate decarboxylase

O 2-dependent oxalate decarboxylase (oxalate carboxy-lyase, EC 4.1.1.2) is isolated of a higher degree of purity. During the decarboxylation reaction, in addition to formate and CO 2, traces of H 2O 2 and oxidation products of aromatic amines and phenols are formed; these aromatic compounds are added with the purpose of stimulating and protecting the enzyme during its catalytic action. When the partial pressure of O 2 is increased the enzyme is denatured at an increasing rate; the amount of H 2O 2 and oxidation products formed are also increased. The results suggest that the oxalate decarboxylation proceeds with a single electron transfer.

Enzymatic determination of oxalic acid-C 14 in plant material

A rapid and accurate manometric method for determining the amount and specific activity of total and water-soluble oxalic acid utilizing oxalic decarboxylase from Collybia velutipes (wood-rot fungus) is described. Two Warburg flasks were used for each determination; in one of them the gas exchange was measured, and in the other the liberated C 14O 2 was trapped on a paper moistened with 1 M Hyamine hydroxide in methanol. The paper was studied in a liquid scintillation counter with high counting efficiency.

Reaction Properties of the Ascorbic Acid Oxidase from Myrothecium verrucaria

Ascorbic acid oxidase activity in Myrothecium verrucaria extracts resulted in O(2) uptake exceeding 0.5 mole per mole of ascorbic acid and in CO(2) evolution. Measurement of oxidized ascorbic acid at completion of the reaction demonstrated that an average of 10% of the oxidized product disappeared. A comparison of the gas exchange data with the amount of ascorbic acid not accounted for indicated that the reaction could not be explained by independent oxidase and oxygenase systems. Chromatographic examination of the reaction mixtures identified l-threonic acid. Experiments with ascorbic acid-1-(14)C showed that C-1 was partially decarboxylated during the oxidation. Test of the fungal extracts for enzymes that might explain the deviation from expected stoichiometry showed that phenolase, glutathione reductase, cytochrome oxidase, peroxidase and oxalic decarboxylase were not involved. Addition of azide in concentrations sufficient to block catalase increased excess O(2) consumption about 65%. No enzymes were found that could directly attack oxidized ascorbic acid. H(2)O(2) accumulated during oxidation in azide-blocked systems.The O(2) excess could be explained by assuming the enzyme had peroxidative capacity on a reductant other than ascorbic acid. An intermediate of ascorbic acid oxidation appeared to function as the substrate yielding CO(2) and l-threonic acid on degradation. The increase in excess O(2) utilized in azide-blocked systems and the H(2)O(2) accumulation also were explained by the proposed scheme.Another interpretation would involve production of free radicals during ascorbic acid oxidation. Evidence for this was the ability of extracts to oxidize DPNH in the presence of ascorbic acid. Oxygen radicals formed in such reactions were considered possible agents of degradation of ascorbic acid.

An oxalic acid decarboxylase of Myrothecium verrucaria

Extracts from spores and mycelium of Myrothecium verrucaria contain an oxalic acid decarboxylase similar to the one Shimazono found in a wood-rotting fungus. The enzyme is highly specific for oxalic acid having no activity on 13 other carboxylic acids including formic acid. The stoichiometry approached 1 mole of oxalic acid per mole of formic acid and carbon dioxide. However, the carbon dioxide yield was about 10% low and there was an average of 0.85 μmole of oxygen consumed per 20 μmoles of oxalic acid decomposed particularly in the later stages of the reaction. The enzyme was sensitive to several heavy metal and sulfhydryl inhibitors. It was inactive under anaerobic conditions and was inhibited by certain reducing agents. The inhibition was reversed by oxygen but not by other electron acceptors. It is suggested that reductive cleavage of certain disulfide bonds inactivates the enzyme and that this process is reversible.

Enzymatic oxalate decarboxylation in Aspergillus niger

Common fungi of the Aspergillus niger group produce an oxygen-dependent oxalate decarboxylase which is especially abundant in those strains that yield greater amounts of citric acid. Oxygen is not consumed during the reaction and causes a denaturation that is proportional to its partial pressure. Certain reducing compounds (e.g., o-phenylenediamine) and proteins (e.g., gelatin) protect and stimulate the purified enzyme when it acts in the presence of air.

Enzymatic Oxalate Determination in Urine

Abstract A method depending upon the use of oxalic decarboxylase is described for the determination of oxalic acid in urine. The urinary oxalate excretion of 25 healthy persons measured by this procedure is found to average 20.5 mg. (COOH)2/24 hr. In 70 patients with calculus disease the oxalate titer of the urine was within the normal range.

A STRAIN OF DESULFOVIBRIO ABLE TO USE OXAMATE.

Strain Monticello 2, a variant of Desulfovibrio desulfuricans, was isolated from an environment associated with Streptococcus allantoicus, which converts allantoin to oxamate. The strain grew with oxamate or oxalate, as well with more conventional substrates for Des. desulfuricans; yeast extract was essential for growth with oxamate, but not for growth with lactate. A cell-free extract formed oxalate quantitatively from oxamate but not from oxamide; it formed a hydroxamate in the presence of hydroxylamine. Unequivocal amidase or transferase activity towards acetamide was not observed. Frozen and thawed organisms decomposed oxalate but a soluble oxalic decarboxylase was not obtained; such preparations did not require ATP; they formed traces of formate from oxalate. The proposition that oxamate is an “incomplete substrate” of ecological value to this strain is discussed.