Atp dependent and ionophore induced proton translocation in pea pisum sativum cultivar alaska stem microsomal vesicles
Vianello, A.; Dell'antone P.; Macri, F.
Biochimica et Biophysica Acta 689(1): 89-96
ISSN/ISBN: 0005-2728 Accession: 004794086
The presence of an electrogenic pump in pea stem microsomal vesicles has already been demonstrated, but no evidence on the nature of the electrogenic ion has been presented. The usefulness of the .DELTA.pH probe, Acridine orange, to monitor ATP-dependent and ionophore-induced H+ fluxes in pea stem microsomal vesicles was tested. The H+/K+ exchanger nigericin causes a marked uptake of protons into the vesicles that can be followed, with similar results, both as Acridine orange absorbance changes and pH changes of the external medium. ATP induces an uptake of Acridine orange into the vesicles which is reversed by FCCP [carbonyl cyanide p-trifluoromethoxyphenylhydrazone] and abolished by the presence of Triton X-100 in the incubation medium, thus indicating an inward, ATP-driven, H+ translocation. The ATP-dependent Acridine orange uptake is Mg2+-requiring and KCl-stimualted. Such activity is inhibited by 2 specific ATPase inhibitors, dicyclohexylcarbodiimide and diethylstilbestrol, while it is unaffected by oligomycin and Na3VO4. These results show that Acridine orange is a useful probe to measure pH gradients in this membrane system and are consistent with the hypothesis that an ATPase of plasmalemma may act as a proton pump.