PLANT PHYSIOLOGY , Vol 106, Issue 1 79-85, Copyright © 1994 by American Society of Plant Biologists
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ENVIRONMENTAL AND STRESS PHYSIOLOGY |
Solubilization and Reconstitution of the Mg2+/2H+ Antiporter of the Lutoid Tonoplast from Hevea brasiliensis Latex
Z. Amalou, R. Gibrat, P. Trouslot and J. d'Auzac
Biochimie et Physiologie Vegetales, Ecole Nationale Superieure Agronomique de Montpellier/Institut National de la Recherche Agronomique/Centre National de la Recherche Scientifique (Unite de Recherche Associee 573), F-34060 Montpellier Cedex 1, France (Z.A., R.G.)
The Mg2+/2H+ antiporter recently described on lutoid membrane (Z. Amalou,
R. Gibrat, C. Brugidou, P. Trouslot, J.d'Auzac [1992] Plant Physiol 100:
255-260) was solubilized by octylglucoside and reconstituted into soybean
liposomes using the detergent dilution method. Magnesium efflux or influx
experiments were used to generate a H+ influx or efflux, respectively,
monitored with the fluorescent probe 9-amino-6-chloro-2-methoxyacridine.
Both experiments gave saturable H+ fluxes as a function of internal or
external Mg2+ concentrations with similar kinetic parameters Km and Vmax.
The Km value for Mg2+ (about 2 mM) was identical to that previously found
in lyophilized-resuspended lutoid (reference therein), whereas the Vmax
value was 14-fold higher. Since only 10% of the initial proteins were
recovered in proteoliposomes, and electrophoretic patterns of the two kinds
of vesicles differed significantly, it was inferred that the increase in
Vmax was due essentially to an enrichment of the protein antiporter in the
reconstituted fraction, owing to a selective effect of octylglucoside at
both solubilization and reconstitution steps. None of the various divalent
cations used could dissipate the pH gradient of control liposomes of
soybean lipids, unless the divalent/H+ exchanger A23187 was added, whereas
a rapid dissipation of the pH gradient was observed with reconstituted
proteoliposomes from lutoid proteins, with the cation selectivity sequence
Zn2+ > Cd2+ > Mg2+ in the millimolar concentration range. The
divalent ions Ca2+, Ba2+, and Mn2+ were incapable of generating a H+ efflux
in reconstituted proteoliposomes, whereas both Mg2+/H+ and Ca2+/H+
exchanges were observed in lyophilized-resuspended lutoids. Therefore, the
lutoid membrane seems to contain separate Mg2+/H+ and Ca2+/H transport
systems, the latter being eliminated during the
solubilization/reconstitution of lutoid membrane proteins.
