The α-Subunit of the Na+/K+-ATPase has Catalytic Activity Independent of the ß-subunit

  • G. Blanco
  • A. W. De Tomaso
  • J. C. Koster
  • Z. J. Xie
  • R. W. Mercer

Abstract

A functional Na+/K+-ATPase enzyme consists of two polypeptides: a large catalytic α-subunit and a smaller glycosylated ß-subunit. Although all catalytic properties of the Na+/K+-ATPase are associated with the α-subunit, it has never been determined if this subunit has activity independent of the ß-subunit. Using the baculovirus expression system we have previously demonstrated that when expressed alone, the rodent α-subunit exists in a stable conformation unassociated with other proteins in the infected Sf-9 cell (3). Here we show that the α-subunit has catalytic activity independent of the ß-subunit. This ATPase activity is dependent on Mg2+, does not require Na+ or K+, and is not inhibited by ouabain. However, the independent α-subunit ATPase activity is inhibited by EGTA, vanadate and increasing ionic strength. The inhibition of the ATPase activity by EGTA is not abolished by the addition of Ca2+ suggesting that EGTA inhibits activity in a manner other than its ion-binding abilities. Phosphorylated intermediates of the independent α-subunit were acid-stable and alkaline-labile, as well as sensitive to hydroxylamine, strongly suggesting the presence of an acylphosphate and thus phosphorylation at an aspartate residue. The physiological role, if any, of this independent α-subunit activity is unknown.

Keywords

Hydrolysis Tyrosine Adenosine Carboxyl Electrophoresis 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. 1.
    Bastide F, Meissner G, Fleischer S, and Post RL (1973) Similarity of the active site of phosphorylation of the adenosine triphosphatase for transport of sodium and potassium ions in kidney to that for transport of calcium ions in the sarcoplasmic reticulum of muscle. J Bio Chem 248: 8385–8391Google Scholar
  2. 2.
    Blanco G, Xie ZJ and Mercer RW (1993) Functional expression of the α2 and α3 isoforms of the Na,K-ATPase in baculovirus infected insect cells. Proc Natl. Acad Sci USA 90: 1824–1828PubMedCrossRefGoogle Scholar
  3. 3.
    De Tomaso AW, Xie ZJ, Liu G and Mercer RW (1993) Expression, targeting and assembly of functional Na,K-ATPase polypeptides in baculovirus-infected insect cells. J Bio Chem 268:1470–11478Google Scholar
  4. 4.
    Duclos B, Marcandier S, and Cozzone AJ (1991) Chemical properties and separation of phosphoamino acids by thin-layer chromatography and/or electrophoresis. Methods Enzymol 201: 10–21PubMedCrossRefGoogle Scholar
  5. 5.
    Hokin LE., Sastry PS, Galsworthy PR, and Yoda A (1965) Evidence that a phosphorylated intermediate in a brain transport adenosine triphosphatase is an acyl phosphate. Proc Natl. Acad Sci USA 54:177–184PubMedCrossRefGoogle Scholar
  6. 6.
    Lipmann F, and Tuttle LC (1945) A specific micromethod for the determination of acyl phosphates. J Bio Chem 159:21–28Google Scholar
  7. 7.
    Post, RL. and Kume S (1973) Evidence for an aspartyl phosphate residue at the active site of sodium and potassium ion transport adenosine triphosphatase. J Bio Chem 248: 6993–7000Google Scholar

Copyright information

© Dietrich Steinkopff Verlag GmbH & Co. KG, Darmstadt 1994

Authors and Affiliations

  • G. Blanco
    • 1
    • 2
  • A. W. De Tomaso
    • 1
    • 2
  • J. C. Koster
    • 1
    • 2
  • Z. J. Xie
    • 1
    • 2
  • R. W. Mercer
    • 1
    • 2
  1. 1.Department of Cell Biology and PhysiologyWashington University School of MedicineSt. LouisUSA
  2. 2.Department of PharmacologyMedical College of OhioToledoUSA

Personalised recommendations