Measurement of Cytoplasmic pH by the DMO Technique inHydrodictyon africanum

The 5, 5-dimethyl-[2-14C]oxazolidine-2, 4-dione (DMO) distribution technique for the measurement of intracellular pH has been applied to giant cells of <it>Hydrodictyon africanum</it>. Significant metabolism of DMO was found in this alga; the free [DMO + DMO−] in subcellular samples is thus derived from the total label in cells equilibrated in [14C]DMO solutions by measuring and subtracting the label in metabolic products of DMO. A further problem arises from the observation that the DMO concentration in the vacuolar sap is always lower than that predicted by the transmembrane equilibration of undissociated DMO from the bathing medium. This is interpreted in terms of a finite permeability to the anion DMO−. Since the effect of P<inf>DMO</inf>− on the DMO distribution is much smaller at the tonoplast (where the transmembrane electrical potential difference is small) than at the plasmalemma, the values of cytoplasmic pH are computed assuming equilibration of undissociated DMO across the tonoplast. At an external pH of 7.0 the cytoplasmic pH is about 7.4; decrease or increase of external pH by 1 unit causes a decrease and an increase in cytoplasmic p11 respectively of about 0.2 pH units. Determinations of Ψ<inf>vo</inf> at pH 6, 7, and 8, together with an assumed constant value of Ψ<inf>cv</inf>, permit calculations of Δμ<inf>H</inf>+ at the plasmalemma and tonoplast. The values are relatively independent of external pH in the range pH 6–8 at 21–25 and 12–14 kJ mol−1 respectively. The significance of these results for the regulation of intracellular pH, and for the regulation and energising of the fluxes of ions, is discussed.