Reconstitution and photolabeling of the purified (Na+ + Mg2+)-ATPase from the plasma membrane of Acholeplasma laidlawii B with phospholipids containing a photosensitive fatty acyl group.
The purified membrane (Na+ + Mg2+)-ATPase of Acholeplasma laidlawii B was reconstituted into vesicles composed of phospholipids containing a photoactivatable aryl nitrene-generating fatty acyl group. The reconstitution with phospholipid resulted in an enhancement of ATPase activity and a reduction in the sensitivity of the enzyme to radiation inactivation. The incorporation of the enzyme into the lipid vesicles results in a broadening of the gel-to-liquid-crystalline phase transition of the photolabeled phospholipid and the appearance of two partially resolved endotherms in the calorimetric traces. The temperatures and the total enthalpy of these overlapping transitions are higher than in the absence of incorporated enzyme. After photolysis of the lipid-reconstituted ATPase and separation of the polypeptide subunits by sodium dodecyl sulfate (SDS) gel electrophoresis, a significant labeling of the alpha-subunit of the enzyme was demonstrated. These results indicate that at least the alpha-subunit of this ATPase must penetrate into or traverse the phospholipid bilayer.
George
R
R
Lewis
R N
RN
McElhaney
R N
RN
D000128:Acholeplasma laidlawii / Q000201:enzymology*; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D008051:Lipid Bilayers / Q000378:metabolism; D046911:Macromolecular Substances; D010713:Phosphatidylcholines / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D010782:Photolysis
D008051:Lipid Bilayers; D046911:Macromolecular Substances; D010713:Phosphatidylcholines; D010743:Phospholipids; C019267:azidophosphatidylcholine; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; C035123:magnesium sodium ATPase