Malonate decarboxylation by Pseudomonas fluorescens. II. Magnesium dependency and trapping of active intermediates.

The procedures for culturing the experimental organism, P. jhorescens, and for preparing the crude cell-free extract have been described previously (1). Partial Purification of Crude Enzyme Preparation-Powdered ammonium sulfate was added to a given volume of the crude cell-free extract to obtain 20 per cent saturation. The resulting precipitate was collected by centrifugation for 30 minutes at 20,000 X g. The supernatant fluid was decanted, and the precipitate was drained by inverting the centrifuge tube on absorbent paper toweling. The precipitate was redissolved in distilled water. The supernatant fluid was then brought to 40 per cent saturation, and the procedures were repeated until solutions of the ammonium sulfate fractions of 0 to 20, 20 to 40, 40 to 60, 60 to 80, and 80 to 100 per cent saturation were obtained. The pH of these resulting solutions ranged from 5.5 to 6.0. All the manipulations were performed in the cold (4’ f 1”) with the exception of the salting out step, which was carried out at room temperature. The nomogram of Dixon (3) was employed for calculating the required amounts of ammonium sulfate. Dialysis-5 ml. of the enzyme solution were placed in a cellophane dialy-