Identification of inositol hexaphosphate in 31P-NMR spectra of Dictyostelium discoideum amoebae. Relevance to intracellular pH determination

Abstract

A sugar phosphomonoester, myo-inositol hexakisphosphate (phytic acid), has been identified as a major phosphorylated metabolite in Dictyostelium discoideum amoeba. Its intracellular concentration was estimated to be 0.7 mM. The identification was made in perchloric acid extracts on the basis of 31P-NMR chemical shift values and their variations with pH, by addition of authentic compound and by hydrolysis with wheat phytase. Perchloric acid extracts were prepared so as to avoid losses of insoluble salts of polyphosphorylated compounds with divalent cations. The glycolytic intermediate, 3-phosphoglycerate accumulated intracellularly in amoebae incubated in the presence of fluoride. The pH sensitive NMR signal of 3-phosphoglycerate was used to monitor cytosolic pH and a value of pH 7.4 was found in aerobic Dictyostelium amoebae.