Figure 10.

Rate of UMP biosynthesis in WT and oligomerization deficient mutant HsUMPS.A and B, LC-MS/MS quantification of isotopically labeled UMP (UMP∗) was used to compare the rate of de novo biosynthesis of UMP in rich media (A) and nucleotide depleted media (B). While the UMP concentrations in cells transfected with plasmid expressing native UMPS (blue circles and line) level off after time in rich media and take time to reach maximal velocity in depleted media, cells transfected with plasmids harboring the oligomerization-deficient mutant (green squares and line) appear to have a consistent, high rate of biosynthesis. C, as a control, the total UMP concentration (C), calculated from a standard curve using an authentic UMP standard, was determined from cells transfected with plasmids containing WT HsUMPS (blue) or the oligomerization deficient (S87TQ90E) mutant (green) over time. The error bars shown represent the 95% confidence interval, and the p-values listed are from independent t-tests comparing the two sets of triplicate values at that time point. HsUMPS, human UMP synthase; OPRT, orotate phosphoribosyl transferase; UMP, uridine 5′-monophosphate.