Purification of the human α2 lsoform of Na,K-ATPase expressed in Pichia pastoris.: Stabilization by lipids and FXYD1

Human alpha 1 and alpha 2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidinetagged beta 1 subunit in Pichia pastoris. Methanol-induced expression of alpha 2 is optimal at 20 degrees C, whereas at 25 degrees C, which is optimal for expression of alpha 1, alpha 2 is not expressed. Detergent-soluble alpha 2 beta 1 and alpha 1 beta 1 complexes have been purified in a stable and functional state. alpha 2 beta 1 shows a somewhat lower Na,K-ATPase activity and higher K0.5K compared to alpha 1 beta 1, while values of K0.5Na and K(m)ATP are similar. Ouabain inhibits both alpha 1 beta 1 (K-0.5 24.6 +/- 6 nM) and alpha 1 beta 1 (K-0.5 102 +/- 14 nM) with high affinity. A striking difference between the isofonns is that alpha 2 beta 1 is unstable. Both alpha 2 beta 1 and alpha 2 beta 1 complexes, prepared in C12E8 with an added phosphatidyl serine, are active, but alpha 2 beta 1 is rapidly inactivated at 0 degrees C. Addition of low concentrations of cholesterol with I-stearoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (SOPS) stabilizes strongly, maintaining alpha 2 beta 1 active up to two weeks at 0 degrees C. By contrast, alpha 1 beta 1 is stable at 0 degrees C without added cholesterol. Both alpha 1 beta 1 and alpha 2 beta 1 complexes are stabilized by cholesterol at 37 degrees C . Human FXYD1 spontaneously associates in vitro with either alpha 1 beta 1 or alpha 2 beta 1, to form alpha 1 beta 1/FXYD1 and alpha 2 beta 1/FXYD1 complexes. The reconstituted FXYD1 protects both alpha 1 beta 1and alpha 2 beta 1 very strongly against thermal inactivation. Instability of alpha 2 is attributable to suboptimal phophatidylserine-protein interactions. Residues within TM8, TM9 and TM10, near the alpha beta subunit interface, may play an important role in differential interactions of lipid with alpha 1 and alpha 2, and affect isoform stability. Possible physiological implications of isoform interactions with phospholipids and FXYD1 are discussed.