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Transamination in Pseudomonas aeruginosa MacQuillan, Anthony Mullens

Abstract

In attempting to study transamination in Pseudomonas aeruginosa, with the object of determining the range of compounds concerned and whether or not more than one enzyme is involved, an accurate, rapid, and generally applicable quantitative procedure for measuring amino acids was necessary. A method involving paper chromatography of reaction mixtures, spraying with ninhydrin and colorimetric measurement of the eluted spots was found and suitably modified. The reaction mixture was amino acid, keto acid, pyridoxal phosphate, water, phosphate buffer and enzyme. The range of activity of the crude cell-free extract was investigated by testing its ability to transaminate from 23 amino compounds to glyoxylate, α-ketoglutarate, oxalacetate and pyruvate. No transamination with pyruvate was observed and very little oxalacetate. The range of transamination with glyoxylate and α-ketoglutarate was extensive. In order to test whether or not these activities were due to one enzyme, purification was attempted. Isoleucine-glutamate was the system whose activity was followed. Partial purification of the enzyme catalyzing this reaction was achieved by precipitating the nucleic acids with protamine sulphate and subsequently fractionating with ammonium sulphate. The isoleucine-glutamate activity was most concentrated in the 50/60 fraction. Further purification of this enzyme system was attempted with the use of calcium phosphate gel adsorption and elution; ion-exchange resin columns; paper electrophoresis in phosphate buffers and ammonium sulphate elution from a celite column - all without success. Having achieved some purification of the isoleucine-glutamate catalyzing system, the range and specificity of this partially purified fraction was compared with that of crude cell-free extract. The results showed that the partially purified fraction retained the broad range of glyoxylate and α-ketoglutarate activities while the range of oxalacetate activity was greatly increased. The possibility of chemical transamination under reaction conditions was examined and it was observed that glyxoylate can be chemically aminated in every case where it was thought that enzymatic transamination might occur. The concentration of other transaminating activities in a number of ammonium sulphate fractions was examined. The systems studied were isoleucine, methionine and phenylalanine, each with α-ketoglutarate, as well as isoleucine phenylanaline and glutamate each with oxalacetate. Results indicated that the activities involving α-ketoglutarate were concentrating in the 50/60 fraction while those involving oxalacetate were concentrating in the 60/70 fraction. Specific activities corroborated these observations to a large extent. These results indicated at least two transaminases in P. aeruginosa. The glyoxylate system was re-examined by quantitative comparison of chemical and enzymatic transamination and also by stopping the reactions with trichloracetic acid rather than by heat. Each of these procedures indicated that glutamate will enzymatically transaminate with glyoxylate to form glycine. Other amino acids tested were inactive. The question of pyruvate participation was investigated and the presence of a glutamate-alanine system was found in fresh, crude preparations. This activity was not shown to occur in the 50/60 fraction. The observed facts therefore suggest the possibility of at least three transaminating systems in P. aeruginosa.

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