Purification and properties of adenylosuccinate synthetase from Yoshida sarcoma ascites tumor cells

Abstract

Adenylosuccinate synthetase (IMP: l-aspartate ligase (GDP-forming), EC 6.3.4.4) was purified about 750-fold to a homogeneous state from Yoshida sarcoma ascites tumor cells. A yield of 38% purified enzyme was achieved by a procedure including affinity chromatography on hadacidin-Sepharose 4B. Ultracentrifugal analyses showed that the molecular weight of the native enzyme was 102 000 with an s 20, w value of 4.5 and that the molecular weight in 6 M guanidine-HCl was 47 000. These values indicate that the native enzyme is composed of two subunits. The isoelectric point was determined to be 5.9 by isoelectric focusing. The optimum pH for activity was 6.8-7.0. The K m values for IMP, aspartate and GTP were calculated to be 4.1, 9.8 and 0.7 · 10 −4 M , respectively. The antibiotic, hadacidin was strongly inhibitory, causing competitive inhibition with respect to aspartate with a K 1 value of 2.5 · 10 −6 M . Nucleoside mono- and diphosphate also inhibited the enzyme activity, but their inhibitions were not apparently specific. The purified enzyme showed full activity in the presence of Mg 2+, and Mg 2+ could be partially replaced by Mn 2+, Co 2+, Ca 2+ or Cu 2+. Divalent metal ions, such as Cd 2+, Pb 2+, Zn 2+, Cu 2+ and Mn 2+, interfered with the activity by antagonizing Mg 2+, Hg 2+ or PCMB inactivated the enzyme, suggesting that an SH-group may be important for activity.