Purification and characterization of GMP synthetase from Yoshida sarcoma ascites cells

GMP synthetase was found in the cytosolic fraction of Yoshida sarcoma ascites cells. However, prolonged centrifugation resulted in precipitation of the enzyme. On sucrose density gradient centrifugation of a crude extract of Yoshida sarcoma ascites cells, a part of this enzyme showed high sedimentability at low ionic strength. On the basis of these observations, GMP synthetase was purified from Yoshida sarcoma ascites cells by means of procedures including centrifugal fractionation. The purified enzyme was shown to be homogeneous on SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel. The molecular weight of the GMP synthetase was estimated to be 78,000 by SDS-polyacrylamide gel electrophoresis and gel filtration on Sephadex G-150. Its isoelectric point was estimated to be 5.6. The Km values of this enzyme for XMP, ATP, and glutamine were calculated to be 4.6, 120, and 300 microM, respectively. Although ammonia could substitute for glutamine as a donor of the amino group, the Km value was as high as 120 mM, indicating that it cannot be considered to be a physiological substrate. This enzyme showed high activity only in the presence of Mg2+, and very low activity in the presence of other divalent cations. Inhibition by nucleoside monophosphates was not significant. The enzyme required reduced sulfhydryl compounds for its activity.