Purification of the Human 2 Isoform of Na,K-ATPase Expressed in Pichia pastoris. Stabilization by Lipids and FXYD1

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• 作者：Yael Lifshitz ; Ekaterina Petrovich ; Haim Haviv ; Rivka Goldshleger ; Daniel M. Tal ; Haim Garty ; Steven J. D. Karlish
• 刊名：Biochemistry
• 出版年：2007
• 出版时间：December 25, 2007
• 年：2007
• 卷：46
• 期：51
• 页码：14937 - 14950
• 全文大小：612K
• 年卷期：v.46,no.51(December 25, 2007)
• ISSN：1520-4995

文摘

Human 1 and 2 isoforms of Na,K-ATPase have been expressed with porcine 10*Histidine-tagged 1 subunit in Pichia pastoris. Methanol-induced expression of 2 is optimal at 20 C, whereas at25 C, which is optimal for expression of 1, 2 is not expressed. Detergent-soluble 21 and 11complexes have been purified in a stable and functional state. 21 shows a somewhat lower Na,K-ATPase activity and higher K_0.5K compared to 11, while values of K_0.5Na and K_mATP are similar.Ouabain inhibits both 11 (K_0.5 24.6 ± 6 nM) and 21 (K_0.5 102 ± 14 nM) with high affinity. Astriking difference between the isoforms is that 21 is unstable. Both 11 and 21 complexes, preparedin C₁₂E₈ with an added phosphatidyl serine, are active, but 21 is rapidly inactivated at 0 C. Additionof low concentrations of cholesterol with 1-stearoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (SOPS)stabilizes strongly, maintaining 21 active up to two weeks at 0 C. By contrast, 11 is stable at 0 Cwithout added cholesterol. Both 11 and 21 complexes are stabilized by cholesterol at 37 C. HumanFXYD1 spontaneously associates in vitro with either 11 or 21, to form 11/FXYD1 and 21/FXYD1 complexes. The reconstituted FXYD1 protects both 11 and 21 very strongly against thermalinactivation. Instability of 2 is attributable to suboptimal phophatidylserine-protein interactions. Residueswithin TM8, TM9 and TM10, near the subunit interface, may play an important role in differentialinteractions of lipid with 1 and 2, and affect isoform stability. Possible physiological implications ofisoform interactions with phospholipids and FXYD1 are discussed.