Methyl phosphonate as a 31P-NMR probe for intracellular pH measurements in Dictyostelium amoebae.
Methyl phosphonate at concentrations up to 20 mM was found to be non toxic towards the growth of Dictyostelium amoebae and the starvation-induced differentiation program. In contrast, phenyl phosphonate at the same concentrations was found to inhibit growth. Methyl phosphonate possessed good 31P-NMR characteristics for use as a pH probe in Dictyostelium. The entry of methyl phosphonate into the cytoplasm required about 150 min of incubation before an equilibrium was reached with added extracellular methyl phosphonate. A semilogarithmic plot of the efflux of methyl phosphonate out of the amoebae was linear as a function of time, and the kinetic first-order constant was k = 0.016 min-1. The kinetic parameters were consistent with an uptake of methyl phosphonate by fluid-phase pinocytosis. The pH calculated from the chemical shift of the internalized methyl phosphonate signal was found to be pH 7.4 in aerobic Dictyostelium amoebae. The intracellular methyl phosphonate environment turned more acidic in response to anaerobiosis (delta pH = 0.8) or in the presence of a weak acid such as propionate. These pH determinations were in agreement with the values of cytosolic pH derived from the chemical shift of Pi. Methyl phosphonate should be a useful probe for pH measurements in 31P-NMR studies of Dictyostelium in situations where the signal of Pi cannot be attributed with certainty.
Satre
M
M
Laboratoire de Biochimie (UA 1130 du CNRS), Fédération des Laboratoires de Biologie, Grenoble, France.
Martin
J B
JB
Klein
G
G
D004023:Dictyostelium / Q000254:growth & development / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D015335:Molecular Probes; D009943:Organophosphorus Compounds / Q000493:pharmacokinetics / Q000494:pharmacology*
D015335:Molecular Probes; D009943:Organophosphorus Compounds; C032627:methylphosphonic acid