Inhibition of adenylosuccinate synthetase and adenylosuccinate lyase from Ehrlich ascites-tumour cells by 6-thioinosine 5′-phosphate

Abstract

The apparent activities of adenylosuccinate synthetase, adenylosuccinate lyase and inosine 5[image]-phosphate dehydrogenase were measured in extracts of Ehrlich ascites tumor cells that had been kept at[long dash]15[degree] for periods up to 93 days. In extracts of fresh cells the activity of the synthetase was greater than that of the dehydrogenase and less than that of the lyase. Adenylosuccinate synthetase from the ascites tumor cells was freed of interfering activities and used in kinetic studies of adenylosuccinate synthesis. At pH 7.5 the Michaelis constant (Km) for guanosine 5[image]-tri-phosphate was 0.1 mM. the Km for L-aspartate was 0.05 mM and the Km for inosine 5[image]-phosphate was 0.03 mM. 6-Thioinosine 5[image]-phosphate was a non-competitive inhibitor of conversion of inosine 5[image]-phosphate into adenylosuccinate; the inhibitor constant (Ki) was 0.2-0-4 mM. Adenylosuccinate lyase of adequate purity for kinetic studies was prepared from the ascites tumor cells. Maximum velocities and apparent Michaelis constants for the conversion of adenylosuccinate into adenosine 5[image]-phosphate in the presence and absence of 6-thioinosine 5[image]-phosphate were measured at 12 pH values from pH 5-64 to 8-38. 6-Thioinosine 5[image]-phosphate was a competitive inhibitor; Ki decreased from 0.8 mM at pH 5.64 to 0.1 mM at pH 8.38. The Km for adenylosuccinate increased from 6 [mu]M to 0.07 mM in the same range. Below pH 8 the increase of maximum velocity (V) with decreasing H+ ion concentration was described by the relationship: [image] where [image] was 11.5 um-moles of adenylosuccinate cleaved/min/mg of protein and[long dash]log K1 was 6-3. Similar results were obtained with adenylosuccinate lyase from yeast. From a comparison of the activities and kinetic parameters of the dehydrogenase, synthetase and lyase in Ehrlich ascites tumor cells it seems likely that the first of these activities is the most sensitive to inhibition by 6-thioinosine 5[image]-phos-phate, a metabolite of the inhibitory drug 6-mercaptopurine.