Abstract
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.
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- Eytan G. D. Use of liposomes for reconstitution of biological functions. Biochim Biophys Acta. 1982 Oct 20;694(2):185–202. doi: 10.1016/0304-4157(82)90024-7. [DOI] [PubMed] [Google Scholar]
- Flatman P. W. Mechanisms of magnesium transport. Annu Rev Physiol. 1991;53:259–271. doi: 10.1146/annurev.ph.53.030191.001355. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Marin B., Marin-Lanza M., Komor E. The protonmotive potential difference across the vacuo-lysosomal membrane of Hevea brasiliensis (rubber tree) and its modification by a membrane-bound adenosine triphosphatase. Biochem J. 1981 Aug 15;198(2):365–372. doi: 10.1042/bj1980365. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nakamura T., Hsu C., Rosen B. P. Cation/proton antiport systems in Escherichia coli. Solubilization and reconstitution of delta pH-driven sodium/proton and calcium/proton antiporters. J Biol Chem. 1986 Jan 15;261(2):678–683. [PubMed] [Google Scholar]
- Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
- Pressman B. C. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. [DOI] [PubMed] [Google Scholar]
- Sandstrom R. P., Cleland R. E. Selective delipidation of the plasma membrane by surfactants : enrichment of sterols and activation of ATPase. Plant Physiol. 1989 Aug;90(4):1524–1531. doi: 10.1104/pp.90.4.1524. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]