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Characterization of the Red Beet Plasma Membrane H⁺-ATPase Reconstituted in a Planar Bilayer System
Donald P. Briskin, Swati Basu and Sarah M. Assmann
Vol. 108, No. 1 (May, 1995), pp. 393-398
Published by: American Society of Plant Biologists (ASPB)
Stable URL: http://www.jstor.org/stable/4276548
Page Count: 6
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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 μM 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 ΔpH 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.
Plant Physiology © 1995 American Society of Plant Biologists (ASPB)