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.