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A study of the glucose oxidizing system of pseudomonas aeruginosa Stewart, J. E. (James Edward)


Cell free extracts of glucose grown cultures of P. aeruginosa were prepared by exposure to sonic vibration. These sonicates were capable of oxidizing glucose-6-phosphate, ribose-5-phosphate, glucose, gluconic acid and gluconolactone. Treatment of the sonicate with (NH₄)₂S0₄ resulted in the formation of precipitates which possessed the ability to oxidize glucose, gluconolactone and gluconic acid. After (NH₄)₂SO₄ treatment neither the supernatant nor the precipitate could oxidize the phosphorylated compounds. Since 30.0% (NH₄)₂SO₄ or centrifugation for one half hour at 25,000X g precipitated the enzymes they were considered to be insoluble. Precipitation with different concentrations of (NH₄)₂S0₄ and MnSO₄ failed to separate the glucose and gluconate enzymes from one another. Extraction with bile salts solubilized the gluconate enzyme, but precipitated the glucose enzyme along with a high proportion of the gluconate system. An increase in concentration of the bile salts destroyed the glucose enzyme while a reduction left a large percentage in solution. The addition of glycine to the sonicate solubilized the enzymes but did not aid in their separation. When added before the sonic treatment, the enzymes became labile to protein precipitants. Ethyl alcohol, dioxane and acetone destroyed glucose oxidizing and gluconate oxidizing enzyme activity while ethyl ether destroyed only the glucose system. The use of a growth substrate other than glucose resulted in the formation of a reduced gluconic acid system, but the results were not uniform in that the gluconate enzyme frequently was very active. The glucose system was sensitive to KCN and NaN₃, and 8-hydroxy-quinoline but not to NaF. The 8-hydroxy-quinoline inhibition could be overcome by Mg⁺⁺. Adenosinetriphosphate, flavine adenine dinucleotide, diphosphopyridinonucleotide, triphosphopyridinonucleotide, had no effect on the glucose oxidizing system. Methylene blue, brilliant cresyl blue and pyocyanin had no ability to act as a hydrogen acceptor. However 2.6 dichlorobenzenoneindophenol stimulated the reaction. When this dye was added in the presence of MgS0₄ a 300.0% increase was noted. The product of the reaction was determined by paper chromatography to be gluconic acid. These data indicate that the glucose dehydrogenase differs from any previously described glucose dehydrogenase and that some unknown hydrogen transport system apparently functions in the transport of electrons to the cytochromes. The data support earlier conclusions that the reaction does not involve phosphorylation.

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