On the decomposition of pyrimidines by bacteria. II. Studies with cell-free enzyme preparations.

N. corallina was grown in thymine or glucose media as previously described (Lara, 1952). The cells were harvested by centrifugation, washed, recentrifuged, and frozen in the centrifuge tubes. This facilitates removal of the cellular material from the tubes. The frozen mass was ground to a powder in a cooled mortar, mixed with alumina (1557 AB Levigated Alumina Powder, Buehler Ltd., Chicago; 2 g of alumina per g wet weight of bacteria), and grinding continued until a paste was obtained (McIlwain, 1948). The operation requires about 5 to 7 minutes. The paste was mixed with 2 ml M/30 potassium phosphate buffer, pH 7, per g of bacteria, and centrifuged in a precooled high speed Sorvall centrifuge, kept in a cold room at 15 C. The resulting clear, straw colored supernatant liquid was used as the enzyme extract. Manometric determinations were carried out by conventional methods (Umbreit et al., 1949). Ammonia was quantitatively determined by the method of Conway (1947). Disappearance of barbituric acid was estimated by measuring the extinction in alkaline solution at 260 mu in a Beckman spectrophotometer. The procedure consisted of mixing 0.2 ml of the solution to be analyzed with 0.2 ml 3 N KOH and diluting to 10 ml. All readings were made against a blank prepared in a similar manner, with 0.3 ml M/30 potassium phosphate buffer, pH 7, in place of the barbiturate solution. Separation of thymine and uracil was achieved by chromatographic analysis according to Marshak and Vogel (1951).