ESTIMATION OF THE INTRACELLULAR FREE ADP CONCENTRATION BY F-19 NMR-STUDIES OF FLUORINE-LABELED YEAST PHOSPHOGLYCERATE KINASE INVIVO

Yeast phosphoglycerate kinase was selectively fluorine-labeled in vivo by inducing enzyme synthesis in stationary phase cells in the presenc e of 5-fluorotryptophan. Inducible expression was obtained using a gal actose-inducible expression vector containing the yeast phosphoglycera te kinase coding sequence. F-19 NMR measurements on intact cells showe d two resolved resonances, from the two tryptophan residues in the pro tein, which underwent reversible changes in chemical shift under diffe rent metabolic conditions. Measurements in vitro showed that the diffe rence in the chemical shifts of these two resonances was dependent on the adenine nucleotide concentration, in particular the MgADP concentr ation. A comparison of the spectra obtained in vitro with those obtain ed from the intact cell indicated that in glucose-fed cells the cytoso lic free MgADP concentration was less than 50 muM, which is significan tly lower than the concentrations measured in whole-cell extracts.