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The Influence of magnesium ions on the metabolism of phenylalanine by Pseudomonas Aeruginosa White, Mary Jacqueline

Abstract

The effect of magnesium ions on the metabolism of phenylalanine by Pa. aeruginosa has been studied. In a growing culture, the efficiency of utilization of phenylalanine by Pa. aeruginosa was increased by raising the concentration of MgCl₂.7H₂0 from 0.01% to 2% and decreased by raising the sulfate ion proportionately in a glycerol-phenylalanine-mineral medium. The optimum concentration of magnesium for growth in this medium was 1% MgCl₂.7H₂0. The influence of various factors on the magnesium activation of phenylalanine oxidation by resting cells grown in a magnesium-deficient medium has been determined. The stimulation by added magnesium ions was found to be decreased by increasing the cell concentration. Oxidation by cells harvested from a medium containing 0.1% MgCl₂.7H₂0 was not stimulated by added magnesium ions. The concentration of added magnesium ions was varied from 5 to 5000 ppm. MgCl₂.7H₂0 and was found to be stimulating at all concentrations tried, although 5000 ppm. was slightly less effective. Magnesium as the sulfate had a variable effect, usually stimulatory but not as much as the chloride. Manganous ions did not appear to replace magnesium ions in the stimulation of phenylalanine oxidation. The oxidation of 1-phenylalanine was also stimulated by added magnesium ions. The 1 isomer required 6 moles of oxygen per mole of substrate whereas dl-phenylalanine required only 4 moles. The effect of added magnesium ions on the oxidation of other amino acids was studied. L-tyrosine and dl-vallne were the only amino acids besides dl-phenylalanine whose oxidation was appreciably stimulated by added magnesium ions. Although the oxidation of dl-alanine was somewhat stimulated, the oxidation of dl-serine and l-glutamic acid was inhibited by added magnesium ions. L-histidine was not oxidized either in the presence or absence of magnesium ions. No structural relationship between those compounds stimulated was evident. The effect of pH on the amount of stimulation by added magnesium ions was determined. Oxidation of phenylalanine increased over a range of pH 6 to pH 8 but the greatest percent stimulation by magnesium was obtained at pH 6. Thiamin and a mixture of pyridoxine-pyridexamine were added to determine if magnesium were stimulating phenylalanine oxidation by acting as an activator of these coenzyme precursors. Although the oxidation of phenylalanine was stimulated by both thiamin and pyridoxine-pyridoxamine, the activation by magnesium ions was no greater in the presence of these substances. Keto fixatives were used to determine if the stimulated reaction occurred before the formation of a keto compound. Arsenite inhibited the oxidation completely both in the presence and in the absence of magnesium. Semicarbazide on the other hand, gave a 50% inhibition, the inhibition being slightly less in the presence of magnesium ions. Cyanide did not inhibit the oxidation at 1 x 10ˉ²M or 1 x 10ˉ³M. Since magnesium ions have been found essential to various phosphorylation mechanism, the possibility of phosphorylation of phenylalanine was suggested. No significant phosphorus uptake was found when phenylalanine was oxidized nor did phorphorus appear to be essential to the oxidation of phenylalanine or stimulation by magnesium ions. Deamination of the substrate was found to be slightly inhibited by magnesium. Since pH had been found to markedly influence the oxidation of phenylalanine and % stimulation by magnesium, it was thought that by a determination of the R. Q. (ratio of CO₂ evolved to oxygen taken up), a difference in the type of reaction occurring under these varying conditions might be found. The R. Q. in the presence of magnesium ions at pH 6 and 7 and in the presence or absence of magnesium ions at pH 8 was found to be greatly increased (about 2 times). This would seem to indicate that under these conditions, reactions involving breaking the carbon chain are favored. An attempt was made to detect the possible intermediate compounds in phenylalanine breakdown by oxidation in the Warburg and by chromatography using the ninhydrin reaction. Neither tyramine, phenylethyl amine nor phenylpyruvate were oxidized at any appreciable rate in the Warburg. These compounds cannot be ruled out as possible intermediates on this basis, however, since impermeability of the cell may prevent their oxidation. However, neither tyramine, phenylethyl amine nor tyrosine could be detected by chromatography. It was therefore concluded either that these compounds are not intermediates in the breakdown of phenylalanine or that more than one method of breakdown is occurring thus increasing the difficulty of determining an intermediate compound. A series of phenyl compounds was tested to determine if magnesium were allowing breakdown of the phenyl ring. However, no oxidation of any of the compounds tested was found to occur.

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