Oxidation of Intermediates of the Tricarboxylic Acid Cycle by Extracts of Azotobacter Agile

Abstract

Cultures (24-hr.) of A. agile 4.4 were introduced into 500-ml. Erlenmeyer flasks containing 100 ml. of Burk''s N-free mineral salts medium with 2% sucrose and incubated for 15 hrs. at 30oC on a rotary shaker. Enzyme prepns. were made by suspending 12 gs. of wet cells in a total vol. of 40 ml. with cold 0.1 [image] KHCO3, pH 8.4, and exposing them for 10 min. in the sonic oscillator cooled by circulating ice water. The prepns. contained 1.4-2.2 mg. biuret Nitrogen/ml. and were tested on the same day as prepd. With whole cells a period for adaptation was needed to oxidize all members of the tricarboxylic acid (TCA) cycle except acetate, which was immediately oxidized, and citrate, which was not oxidized even after 1 hr. However, extracts from these cells immediately oxidized all the TCA intermediates tested. The dialyzed extracts had to be supplemented with certain coenzymes for max. oxidation rates with citrate, aketoglutarate and acetate. Citrate was slowly oxidized in the presence of 500 [mu]g. diphosphopyridine nucleotide (DPN), but with the addition of 4 [mu]g. triphospho-pyridine nucleotide (TPN), the Qo2 (N) was increased almost 3-fold, and 16 [mu]g. TPN caused a slightly higher rate of oxidation. It was suggested that the isocitric dehydrogenase of A. agile was TPN-linked. Coenzyme A also stimulated oxidation of citrate, but this was believed due to CoA requirement for oxidation of a-ketoglutarate, considered the product of citrate oxidation. The oxidation rate of a-ketoglutarate was increased by DPN, CoA, and Mg++, but not affected by adenosine triphosphate, cocarboxylase, or protogen. With 50 [mu]g. DPN, the Qo2 (N) was only 40% of that obtained with 500 [mu]g. DPN per vessel. Mg++ had a stimulatory effect on rate of oxidation. Acetate was not oxidized to any extent by cell-free extracts, but was "sparked" by the addition of succinate (1 [mu][image]). All these results support the conclusion that a TCA cycle functions in A. agile 4.4 as was previously established for A. vinelandii.