Stabilization of Na(+),K(+)-ATPase purified from Pichia pastoris membranes by specific interactions with lipids.
Na+,K+-ATPase (porcine alpha1/His10*beta1 or human alpha1/porcine His10*beta1) 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 beta-d-maltoside is an alpha1/beta1 protomer, whereas that in octaethylene glycol dodecyl monoether contains a mixture of alpha1/beta1 protomer and higher order oligomers. The Na+,K+-ATPase activity (8-16 (mumol/min)/mg of protein) is similar in both detergents. Thus, the minimal functional unit is the alpha1/beta1 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 >or= 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 degrees C, but not at 0 degrees 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 alpha1/beta1 subunit interface, thus stabilizing the protein. These interactions do not seem to affect Na+,K+-ATPase activity.
Haviv
Haim
H
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
Cohen
Eytan
E
Lifshitz
Yael
Y
Tal
Daniel M
DM
Goldshleger
Rivka
R
Karlish
Steven J D
SJ
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology*; D002784:Cholesterol / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D015870:Gene Expression; D006801:Humans; D008563:Membrane Lipids / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D010743:Phospholipids / Q000378:metabolism; D010843:Pichia / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D011485:Protein Binding; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013552:Swine
D003902:Detergents; D008563:Membrane Lipids; D010743:Phospholipids; D021122:Protein Subunits; D011994:Recombinant Proteins; D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase