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. K,,, values for IMP, GTP, and L-aspartate are 2 X 1O-4 M, 1 X lop5 M, and 3 X 10e4 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 phenylmethanesulfonyl fluoride, but is completely destroyed by freezing or by 18-hour incubation with 5,5’-dithiobis(2nitrobenzoic acid). Adenylosuccinate synthetase activity has also been found in crude extracts of acetone powders of rabbit heart, liver, kidney, brain, and lung.