Estimation of the intracellular free ADP concentration by 19F NMR studies of fluorine-labeled yeast phosphoglycerate kinase in vivo.
Yeast phosphoglycerate kinase was selectively fluorine-labeled in vivo by inducing enzyme synthesis in stationary phase cells in the presence of 5-fluorotryptophan. Inducible expression was obtained using a galactose-inducible expression vector containing the yeast phosphoglycerate kinase coding sequence. 19F NMR measurements on intact cells showed two resolved resonances, from the two tryptophan residues in the protein, which underwent reversible changes in chemical shift under different metabolic conditions. Measurements in vitro showed that the difference in the chemical shifts of these two resonances was dependent on the adenine nucleotide concentration, in particular the MgADP concentration. A comparison of the spectra obtained in vitro with those obtained from the intact cell indicated that in glucose-fed cells the cytosolic free MgADP concentration was less than 50 microM, which is significantly lower than the concentrations measured in whole-cell extracts.
Williams
S P
SP
Department of Biochemistry and Molecular Biology, University of Manchester, England.
Fulton
A M
AM
Brindle
K M
KM
D000227:Adenine Nucleotides / Q000494:pharmacology; D000244:Adenosine Diphosphate / Q000032:analysis*; D003600:Cytosol / Q000737:chemistry*; D004163:Diphosphoglyceric Acids / Q000494:pharmacology; D004790:Enzyme Induction; D005462:Fluorine Radioisotopes; D005988:Glyceric Acids / Q000494:pharmacology; D006651:Histocytochemistry; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D010735:Phosphoglycerate Kinase / Q000187:drug effects / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014170:Transformation, Genetic; D014364:Tryptophan / Q000031:analogs & derivatives / Q000378:metabolism
D000227:Adenine Nucleotides; D004163:Diphosphoglyceric Acids; D005462:Fluorine Radioisotopes; D005988:Glyceric Acids; D008024:Ligands; D011994:Recombinant Proteins; C015891:glycerate 1,3-biphosphate; C026927:5-fluorotryptophan; D000244:Adenosine Diphosphate; C005156:3-phosphoglycerate; D014364:Tryptophan; D010735:Phosphoglycerate Kinase