The incorporation of acetate in acids of the citric acid cycle by Azotobacter extracts.

Experiments were carried out in manometric vessels of sufficient size to accommodate 18 ml. of reaction mixture. These were made up by using the same proportion of reactants as previously described (1) : 1.20 ml. of 0.2 M phosphate buffer (pH 6.89, 0.6 ml. of 0.02 M MgSOd, 0.6 ml. of 0.025 per cent coenzyme A, 0.6 ml. of 0.1 M sodium acetate-1-C14, 1.2 ml. of 0.1 M sodium citrate, and 3.0 ml. of Azotobacter extract containing approximately 2.5 mg. of N per ml. and prepared by wet crushing with a Booth-Green mill (2). The contents of the flasks were diluted to 18 ml. with water and respiration was measured manometrically at 33” in the usual manner. At time intervals of 0, 20, and 30 minutes, a flask was removed for analysis. The enzymatic reaction was stopped by the addition of HsS04 to pH 1.5, the precipitated protein was removed by centrifugation, and the organic acids were determined by partition chromatography on silica gel by the method of Isherwood (3) as modified by Zbinovsky (4). The various acid fractions were collected, oxidized to COz, and counted as BaC03 to determine the specific radioactivity. All the figures representing significant activity were calculated from samples ranging from 1500 to 8000 counts per 15 minute interval compared to a background of 355 f 23 counts. Geometry and self-absorption corrections were applied.