Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase.
Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca(2+)-ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca(2+) binding sites remain vacant. Moreover, in the presence of Ca(2+), amphipol/detergent mixtures stabilized concentrated ATPase against inactivation and aggregation, whether in the presence or absence of lipids, for much longer periods of time (days) than detergent alone. Our observations suggest that mixtures of amphipols and detergents are promising media for handling solubilized Ca(2+)-ATPase under conditions that would otherwise lead to its irreversible denaturation and/or aggregation.
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Unité de Recherche Associée 2096 (CNRS et CEA) and Section de Biophysique des Protéines et des Membranes, Département de Biologie Cellulaire et Moléculaire, Commissariat à l'Energie Atomique Saclay, 91191 Gif-sur-Yvette Cedex, France.
Menguy
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Tribet
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Popot
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le Maire
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D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D003902:Detergents / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D010316:Particle Size; D011108:Polymers / Q000378:metabolism*; D011489:Protein Denaturation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility
D003902:Detergents; D011108:Polymers; D000252:Calcium-Transporting ATPases