Purification of the human alpha2 Isoform of Na,K-ATPase expressed in Pichia pastoris. Stabilization by lipids and FXYD1.
Human alpha1 and alpha2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged beta1 subunit in Pichia pastoris. Methanol-induced expression of alpha2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha1, alpha2 is not expressed. Detergent-soluble alpha2beta1 and alpha1beta1 complexes have been purified in a stable and functional state. alpha2beta1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha1beta1, while values of K0.5Na and KmATP are similar. Ouabain inhibits both alpha1beta1 (K0.5 24.6 +/- 6 nM) and alpha2beta1 (K0.5 102 +/- 14 nM) with high affinity. A striking difference between the isoforms is that alpha2beta1 is unstable. Both alpha1beta1 and alpha2beta1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha2beta1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-l-serine] (SOPS) stabilizes strongly, maintaining alpha2beta1 active up to two weeks at 0 degrees C. By contrast, alpha1beta1 is stable at 0 degrees C without added cholesterol. Both alpha1beta1 and alpha2beta1 complexes are stabilized by cholesterol at 37 degrees C. Human FXYD1 spontaneously associates in vitro with either alpha1beta1 or alpha2beta1, to form alpha1beta1/FXYD1 and alpha2beta1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha1beta1 and alpha2beta1 very strongly against thermal inactivation. Instability of alpha2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alphabeta subunit interface, may play an important role in differential interactions of lipid with alpha1 and alpha2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot, 76100, Israel.
Yael
Y
Lifshitz
Ekaterina
E
Petrovich
Haim
H
Haviv
Rivka
R
Goldshleger
Daniel M
DM
Tal
Haim
H
Garty
Steven J D
SJ
Karlish
D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002784:Cholesterol / Q000494:pharmacology*; D015870:Gene Expression; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D010718:Phosphatidylserines / Q000494:pharmacology*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011489:Protein Denaturation; D020836:Protein Structure, Quaternary; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013696:Temperature
D007527:Isoenzymes; D008565:Membrane Proteins; D010718:Phosphatidylserines; D010750:Phosphoproteins; C519327:stearoyl-oleoyl-phosphatidylserine; C069141:phospholemman; D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase