Galactose 1 phosphate

For quantification of sugar-phosphate molecules, samples were grown in minimal medium containing 1% (wt/vol) uniformly labeled ¹³C-glucose (Cambridge Isotopes) to early exponential phase at 30°C. A time-zero sample was taken for metabolite profiling as described above. Next, either 1,2-¹³C galactose (Cambridge Isotopes) or 1,2-¹³C fructose (Cambridge Isotopes) was added to each culture to a final concentration of 1% (wt/vol). After 2 h, samples were taken for metabolite extraction. Sample were prepared as described above, and the dried metabolite samples were resuspended in HPLC-grade water containing 5 μg/ml (wt/vol) either unlabeled galactose-1-phosphate (Sigma-Aldrich) or fructose-1-phosphate (BOC Sciences). Sugar-phosphate levels were measured using method 1 as described above. Concentrations were calculated by determining the ratio of peak areas for the endogenously produced 1,2-¹³C-labeled peak and the unlabeled standard peak from the same injection. This experimental design avoids inaccurate quantification due to other potentially confounding hexose phosphates present in yeast cells. The intracellular concentration of each sugar-phosphate was calculated assuming an average cell volume of 45 fl.