Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer |
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Authors: | Françoise Simon-Plas Kees Venema Jean-Pierre Grouzis Rémy Gibrat Jacqueline Rigaud Claude Grignon |
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Affiliation: | (1) Biochimie et Physiologie Végétales, Institut National de la Recherche Agronomique, Centre National de la Recherche Scientifique (URA 573), Ecole Nationale Supérieure Agronomique, 34060 Montpellier Cedex 1, France |
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Abstract: | Summary The purified (H+ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H+-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H+)ATPase molecules were closely associated with liposomes, exhibiting a high H+-pumping activity (150,000% quenching· min–1·mg–1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H+)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H+)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles (inside-out orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H+)ATPase could also functionally insert into bilayer from PCPEPG or PCPEPI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H+)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H+)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation |
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Keywords: | (H+)ATPase plant plasma membrane spontaneous insertion liposomes corn root |
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