Stabilization of Na+,K+-ATPase Purified from Pichia pastoris Membranes by Specific Interactions with Lipids

Na+,K+-ATPase (porcine α1/His10*β1 or human α1/porcine His10*β1) has been expressed in Pichia pastoris and purified by Co2+-chelate affinity resin chromatography, yielding about 80% pure, functional, and stable protein in a single step. The protein was eluted in nonionic detergents together with a phosphatidylserine. Size exclusion chromatography showed that the protein eluted in n-dodecyl β-d-maltoside is an α1/β1 protomer, whereas that in octaethylene glycol dodecyl monoether contains a mixture of α1/β1 protomer and higher order oligomers. The Na+,K+-ATPase activity (8−16 (μmol/min)/mg of protein) is similar in both detergents. Thus, the minimal functional unit is the α1/β1 protomer, and activity is unaffected by the presence of oligomeric forms. Screening of phospholipids for stabilization of the Na+,K+-ATPase activity shows that (a) acid phospholipids are required and phosphatidylserine is somewhat better than phosphatidylinositol and (b) optimal stabilization is achieved with asymmetric phosphatidylserines having saturated (18:0 ≥ 16:0) and unsaturated (18:1 > 18:2) side chains at sn-1 an sn-2 positions, respectively. In the presence of phosphatidylserine, cholesterol stabilizes the protein at 37 °C, but not at 0 °C. Cholesterol also increases the “apparent affinity” of the phosphatidylserine and stabilizes optimally in the presence of phosphatidylserines with a saturated fatty acyl chain at the sn-1 position. Ergosterol is a poor stabilizer. We propose that phosphatidylserine and cholesterol interact specifically with each other near the α1/β1 subunit interface, thus stabilizing the protein. These interactions do not seem to affect Na+,K+-ATPase activity.