Estimation of cytoplasmic free Mg²⁺ levels and phosphorylation potentials in mung bean root tips by in vivo ³¹P NMR spectroscopy

The cytoplasmic [MgATP]/[ATP]_free ratios, free Mg²⁺ concentrations, and phosphorylation potentials in mung bean [Vigna mungo (L.) Hepper] root tip cells were investigated by ³¹P nuclear magnetic resonance spectroscopy. ³¹P NMR spectra show well defined peaks due to G6P, cytoplasmic P_i, vacuolar P_i, ATP, UDP-glucose and nicotinamide adenine nucleotides. The concentrations of phosphorus metabolites were determined from quantitative ³¹P NMR spectra. The [MgATP]/[ATP]_free ratio was 9.45. Accordingly, about 90% of the cytoplasmic ATP was complexed to Mg²⁺. Utilizing the dissociation constant (K_d) determined for MgATP, the cytoplasmic free Mg²⁺ concentration was estimated to be 0.4mM. The NMR-derived phosphorylation potential, [ATP]/([ADP][P_i]), was 960 M^-1. The sodium azide treatment decreased the [ATP]/[ADP] ratio and the phosphorylation potential, and increased the [Mg²⁺]^free. Metabolic inhibition may have been enhanced by an increase in [Mg^2+free and a decrease in the free energy change for ATP hydrolysis, which resulted due to a decrease in the ATP level.