PLANT PHYSIOLOGY , Vol 108, Issue 1 393-398, Copyright © 1995 by American Society of Plant Biologists
Characterization of the Red Beet Plasma Membrane H+-ATPase Reconstituted in a Planar Bilayer System
D. P. Briskin, S. Basu and S. M. Assmann
Department of Agronomy, University of Illinois, Urbana, Illinois 61801 (D.P.B., S.B.)
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.
