Reconstitution of the purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes into lipid vesicles and a characterization of the resulting proteoliposomes.
The purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes was reconstituted with dimyristoylphosphatidylcholine using a cholate solubilization and dialysis procedure. The incorporation of this enzyme into the phospholipid bilayer is accompanied by an enhancement of its specific activity and by a restoration of its lipid phase state-dependent properties which were lost during solubilization and purification from native membranes. Moreover, reconstitution of this ATPase with phospholipid also stabilizes it against cold inactivation at low temperatures (approximately equal to 0 degrees C), oxidative degradation at room temperature, and thermal denaturation at elevated temperatures (approximately equal to 55 degrees C). The elution profile from a Sepharose 4B-CL column indicates that all of the ATPase protein is associated with the phospholipid vesicles and that the Stoke's radius of the proteoliposomes formed is smaller than that of the lipid vesicles formed in the absence of any protein. The latter conclusion is supported by sedimentation velocity measurements and by an electron microscopic examination of negatively stained preparations. The electron microscopic studies demonstrate that sealed vesicles are formed only at low protein-to-lipid ratios. These observations indicate that the Acholeplasma laidlawii B (Na+ + Mg2+)-ATPase has been structurally and functionally reconstituted into lipid vesicles and that the proteoliposomes formed are amenable to studies aimed at the clarification of its proposed role as a sodium ion pump.
D000128:Acholeplasma laidlawii / Q000201:enzymology*; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002850:Chromatography, Gel; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D004355:Drug Stability; D008051:Lipid Bilayers / Q000378:metabolism; D008081:Liposomes / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D013696:Temperature; D014461:Ultracentrifugation
D008051:Lipid Bilayers; D008081:Liposomes; D008563:Membrane Lipids; D008565:Membrane Proteins; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C035123:magnesium sodium ATPase; D004134:Dimyristoylphosphatidylcholine