Open AccessCharacterization of the Red Beet Plasma Membrane H+-ATPase Reconstituted in a Planar Bilayer System
Author(s) -
Donald P. Briskin,
Swati Basu,
Sarah M. Assmann
Publication year - 1995
Publication title -
plant physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.554
H-Index - 312
eISSN - 1532-2548
pISSN - 0032-0889
DOI - 10.1104/pp.108.1.393
Subject(s) - bilayer , membrane , chemistry , atpase , atp hydrolysis , biophysics , vanadate , analytical chemistry (journal) , biochemistry , enzyme , chromatography , biology
The transport activity of the red beet (Beta vulgaris L.) plasma membrane H+-ATPase was examined following reconstitution into a planar bilayer membrane. Fusion of partially purified plasma membrane H+-ATPase with the bilayer membrane was accomplished by perfusion of proteoliposomes against the bilayer under hypoosmotic conditions. Following incorporation into the bilayer, an ATP-dependent current was measured that demonstrated properties consistent with those of the plasma membrane H+-ATPase. Current production was substrate specific for ATP, inhibited by orthovanadate, and insensitive to 200 nM erythrosin B but inhibited by 100 [mu]M erythrosin B. When current production was measured as a function of Mg:ATP concentration, a simple Michaelis-Menten relationship was observed and a Km of 0.62 mM was estimated. Current-voltage analysis of ATP-dependent current in the presence of 0.5 mM ATP, 20 mM ADP, 40 mM orthophosphate, and an opposing 2.5-unit [delta]pH revealed a reversal potential of about -149 mV. Based on the free energy available from ATP hydrolysis, this reversal potential is consistent with an H+/ATP stoichiometry of 1. This study demonstrates the usefulness of a planar bilayer system for investigation of energy coupling to H+ transport by the plasma membrane H+-ATPase.