Structure of the Cation Occlusion Domain of Na+/K+ ATPase Studied by Proteolysis, Interaction with Ca2+ ions and Thermal Denaturation

  • A. M. Shainskaya
  • S. J. D. Karlish


Knowledge of the structure of cation binding sites and of organization of transmembrane segments of cation pumps, such as Na+/K+-ATPase, is essential for an understanding of the active transport mechanism. Renal Na+/K+-ATPase extensively digested with trypsin, in the presence of Rb+ and absence of Ca2+ ions, to a Cterminal 19kDa and smaller membrane-embedded fragments (8–11kDa) of the alpha chain, and intact beta chain or beta split into two fragments, is a valuable tool for establishing structure-function relationships. In these “19kDa membranes”, cation occlusion sites are intact, and although it is evident that the sites are located within trans-membrane segments, the identity and number of segments involved is unknown. The question as to which or how many of the trans-membrane segments actually occlude the ions is unanswered, and is the major focus of the present work. The first section of the present paper analyses the nature of the antagonism between Ca2+ and Rb+ ions in “19kDa-membranes”, and explains the previous observation that the presence of Ca2+ during digestion destabilises the 19kDa fragment to trypsin and occlusion is destroyed. The second section describes the purification and identification of the limit tryptic membrane-embedded fragments, produced by digestion of “19kDa-membranes” in the presence of Ca2+.


Transmembrane Segment Thermal Inactivation 19kDa Fragment Alpha Chain 19kDa Membrane 
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  1. 1.
    Capasso JM, Hoving S, Tal DM, Goldshleger R, and Karlish SJD (1992) Extensive digestion of Na+,K+-ATPase by Specific and Non-specific Proteases with Preservation of Cation Occlusion sites. J Biol. Chem 267: 1150–1158PubMedGoogle Scholar
  2. 2.
    Carafoli E (1992) The Ca2+ pump of the plasma membrane. J Biol. Chem 267: 2115–2118PubMedGoogle Scholar
  3. 3.
    David P, Mayan H, Cohen H, Tal D, and Karlish SJD (1992) Guanidinium Derivatives act as High Affinity Antagonists of Na+ ions in Occlusion sites of Na+,K+-ATPase. J Biol. Chem 267: 1141–1149PubMedGoogle Scholar
  4. 4.
    Forbush B, III (1988) Rapid Release of 45Ca from an Occluded State of the Na+,K+-ATPase. J Biol. Chem 236: 7970–7978Google Scholar
  5. 5.
    Goldshleger R, Shachak Y, and Karlish SJD (1990) Electrogenic and electroneutral Transport of Renal Na/K-ATPase Reconstituted into Proteoliposomes. J Membr Biol. 113: 139–154PubMedCrossRefGoogle Scholar
  6. 6.
    Goldshleger R, Tal D, Moorman J, Stein WD, and Karlish SJD (1992) Chemical Modification of Glu 953 of the Alpha Chain of Na+,K+-ATPase Associated with Inactivation of Cation Occlusion. Proc Natl. Acad Sci U.S.A. 89: 6911–6915PubMedCrossRefGoogle Scholar
  7. 7.
    Karlish SJD, Goldshleger R, and Stein WD (1990) Identification of a 19kDa Cterminal Tryptic Fragment of the Alpha Chain of Na+,K+-ATPase, Essential for Occlusion and Transport. Proc Natl. Acad Sci U.S.A. 87: 4566–4570PubMedCrossRefGoogle Scholar
  8. 8.
    Karlish SJD, Goldshleger R, and Jørgensen PL (1993) Location of Asn-831 of the Alpha Chain of Na/K-ATPase at the Cytoplasmic Surface. Implication for Topological Models. J Biol. Chem 268: 3471–3478PubMedGoogle Scholar
  9. 9.
    Or E, David P, Shainskaya A, Tal DM, Karlish SJD (1993) Effects of Competitive Sodium-like Antagonists on Na/K-ATPase Suggest that Cation Occlusion from the Cytoplasmic Surface Occurs in Two Steps. J Biol. Chem 268: 16929–16937PubMedGoogle Scholar
  10. 10.
    Ovchinnikov YuA, Arzamazova NM, Arystarkhova EA, Gevondyan NM, Aldanova NA, and Modyanov NN (1987) Detailed structural analysis of exposed domains of membrane bound Na+,K+-ATPase. A model for trans-membrane arrangement. FEBS Letters 217: 269–274PubMedCrossRefGoogle Scholar
  11. 11.
    Palmgren GM, Sommarin M, Serrano R, and Larson Ch (1991) Identification of an Auto inhibitory Domain in the C-terminal Region of the Plant Plasma Membrane H+-ATPase. J Biol. Chem 266: 20470–20475PubMedGoogle Scholar
  12. 12.
    Post RL, and Stewart HB (1985) Occupancy of a monovalent cation-binding centre in Na+,K+-ATPase by calcium ion. In: Glynn I, Ellory C (eds) The Sodium Pump. The Company of Biologists, Cambridge, pp. 429–441Google Scholar
  13. 13.
    Shani-Shekler M, Goldshleger R, Tal D, and Karlish SJD (1988) Inactivation of Rb+ and Na Occlusion on (Na,K)-ATPase by modification of carboxyl groups. J Biol. Chem 263: 19331–19341Google Scholar
  14. 14.
    Vassalo PM, Post RL (1986) Calcium Ion as a Probe of the Monovalent Centre of Sodium Potassium ATPase. J Biol. Chem 261: 16957–16962Google Scholar

Copyright information

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

Authors and Affiliations

  • A. M. Shainskaya
    • 1
  • S. J. D. Karlish
    • 1
  1. 1.Biochemistry DepartmentWeizmann Institute of ScienceRehovotIsrael

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