Purification of Na+,K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions

Na+, K+-ATPase ( porcine alpha/his(10)-beta) has been expressed in Pichia Pastoris, solubilized in n-dodecyl-beta-maltoside and purified to 70 - 80% purity by nickel-nitrilotriacetic acid chromatography combined with size exclusion chromatography. The recombinant protein is inactive if the purification is done without added phospholipids. The neutral phospholipid, dioleoylphosphatidylcholine, preserves Na+, K+-ATPase activity of protein prepared in a Na+-containing medium, but activity is lost in a K+-containing medium. By contrast, the acid phospholipid, dioleoylphosphatidylserine, preserves activity in either Na+- or K+-containing media. In optimal conditions activity is preserved for about 2 weeks at 0 degrees C. Both recombinant Na+, K+-ATPase and native pig kidney Na+, K+-ATPase, dissolved in n-dodecyl-beta-maltoside, appear to be mainly stable monomers (alpha/beta) as judged by size exclusion chromatography and sedimentation velocity. Na+, K+-ATPase activities at 37 degrees C of the size exclusion chromatography-purified recombinant and renal Na+, K+-ATPase are comparable but are lower than that of membrane-bound renal Na+, K+-ATPase. The beta subunit is expressed in Pichia Pastoris as two lightly glycosylated polypeptides and is quantitatively deglycosylated by endoglycosidase- H at 0 degrees C, to a single polypeptide. Deglycosylation inactivates Na+, K+-ATPase prepared with dioleoylphosphatidylcholine, whereas dioleoylphosphatidylserine protects after deglycosylation, and Na+, K+-ATPase activity is preserved. This work demonstrates an essential role of phospholipid interactions with Na+, K+-ATPase, including a direct interaction of dioleoylphosphatidylserine, and possibly another interaction of either the neutral or acid phospholipid. Additional lipid effects are likely. A role for the beta subunit in stabilizing conformations of Na+, K+-ATPase ( or H+, K+-ATPase) with occluded K+ ions can also be inferred. Purified recombinant Na+, K+-ATPase could become an important experimental tool for various purposes, including, hopefully, structural work.