[23] Phosphoribosylglycinamide synthetase from Aerobacter aerogenes

Publisher Summary This chapter describes the purification procedure for phosphoribosylglycinamide synthetase from Aerobacter aerogenes organism. Phosphoribosylamine: glycine ligase [ADP] (PRG) synthetase catalyzes in the de novo path of purine biosynthesis in bacteria as well as higher organisms. The chapter discusses the preparation procedure from Aerobacter aerogenes or, according to the current classification of the original strain, Klebsiella pneurnoniae . Two assays are described. The colorimetric assay is recommended for enzyme purification. It is fast, sensitive, and works well with crude enzyme preparations. The second––radioisotopic assay––is more readily quantitated. It does not work well with crude enzyme preparations. The measurement is carried out in two steps: the formation of PRG and the PRG assay. The purification is carried out at 3° unless otherwise stated. Crude cell extracts are stable for one day of storage on ice, and enzyme at the intermediate stages of purification is stable for several days.