Purification of Adenylosuccinate Synthetase from Rabbit Skeletal Muscle

Adenylosuccinate synthetase has been purified more than 250-fold from extracts of rabbit muscle acetone powder by heating to 60°, ammonium sulfate fractionation, phosphocellulose and hydroxylapatite chromatography, and Sephadex G-150 gel filtration. Crystals have been obtained by dialysis against 60 to 67% ammonium sulfate. Enzyme activity is stable for about 2 weeks in phosphate buffer containing 1 mm dithiothreitol; for longer periods, dialysis against saturated ammonium sulfate is preferable. The enzyme is quite basic, has a molecular weight of approximately 54,000, and does not appear to dissociate into subunits upon treatment with sodium dodecyl sulfate or mercaptoethanol. A broad maximum in enzyme activity occurs at pH ∼ 6.6, and the kinetic parameters of the enzyme differ significantly from those of the Escherichia coli or Ehrlich ascites-tumor cell enzymes. Km values for IMP, GTP, and l-aspartate are 2 x 10-4 m, 1 x 10-5 m, and 3 x 10-4 m, respectively, for the muscle enzyme. GDP, AMP, adenylosuccinate, argininosuccinate, phosphate, arsenate, and sulfate inhibit enzyme activity. Enzyme activity is unaffected by incubation for 24 hours with EDTA, azide, and phenyl-methanesulfonyl fluoride, but is completely destroyed by freezing or by 18-hours incubation with 5,5'-dithiobis(2-nitrobenzoic acid). Adenylosuccinate synthetase activity has also been found in crude extracts of acetone powders of rabbit heart, liver, kidney, brain, and lung.