title: Purification of Na+,K+-ATPase expressed in Pichia pastoris reveals an essential role of phospholipid-protein interactions.
abstract: Na+,K+-ATPase (porcine alpha/his10-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.
authors.affiliation: Department of Biological Chemistry, Weizmann Institute of Science, Rehovoth, Israel.
authors.forename: Eytan
authors.initials: E
authors.lastname: Cohen
authors.forename: Rivka
authors.initials: R
authors.lastname: Goldshleger
authors.forename: Alla
authors.initials: A
authors.lastname: Shainskaya
authors.forename: Daniel M
authors.initials: DM
authors.lastname: Tal
authors.forename: Christine
authors.initials: C
authors.lastname: Ebel
authors.forename: Marc
authors.initials: M
authors.lastname: le Maire
authors.forename: Steven J D
authors.initials: SJ
authors.lastname: Karlish
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D001671:Biochemistry / Q000379:methods; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D003470:Culture Media / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D005960:Glucosides / Q000737:chemistry; D006031:Glycosylation; D007477:Ions; D007668:Kidney / Q000378:metabolism; D013058:Mass Spectrometry; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives* / Q000737:chemistry; D009942:Organometallic Compounds / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry / Q000378:metabolism; D010743:Phospholipids / Q000737:chemistry; D010843:Pichia / Q000201:enzymology*; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry* / Q000302:isolation & purification*; D013552:Swine; D013696:Temperature; D013997:Time Factors
chemical_list: D003470:Culture Media; D005960:Glucosides; D007477:Ions; D009942:Organometallic Compounds; D010713:Phosphatidylcholines; D010718:Phosphatidylserines; D010743:Phospholipids; D011994:Recombinant Proteins; C088321:nickel nitrilotriacetic acid; C040358:dodecyl maltoside; C042511:1,2-dioleoylphosphatidylserine; D000251:Adenosine Triphosphatases; D000254:Sodium-Potassium-Exchanging ATPase; C017251:1,2-oleoylphosphatidylcholine; D009571:Nitrilotriacetic Acid; D011188:Potassium