V-51 NMR investigation of cell-associated vanadate species in Phycomyces blakesleeanus mycelium

Abstract

V-51 NMR spectroscopy was used for detection and identification of cell-associated vanadate (V5+) species after exposure of Phycomyces blakesleeanus mycelium, in exponential phase of growth, to sodium orthovanadate. Complete disappearance of monomer and dimer signals and decreased intensity of the tetramer signal were observed about 40 min after treatment. Simultaneously, a signal at -532 ppm, with increasing intensity, was detected in spectra. The time-dependent rise in this signal was connected to a decrease in the extracellular monomer signal, indicating its transport into the cell. The signal at -532 ppm did not belong to any known simple oxido-vanadate species, nor to a complex with any of the components of experimental medium. This signal was the only one present in spectrum of the mycelium washed 35 min after treatment, and the only one observed in mycelium cultivated on vanadate-contained medium. Therefore, its appearance can be attributed to intracellular complexation, and may represent an important detoxification mechanism of the cell exposed to a physiologically relevant concentration of vanadate. Experiments (V-51 NMR and polarography) performed with Cd-pretreated mycelium (inhibitor of an enzyme responsible for V5+ reduction) and ferricyanide-preincubated mycelium excluded the possibility of V5+ tetramer's entry into the cell.