The Structure of (Na+,K+)‐ATPase as Revealed by Electron Microscopya

Mohraz, Manijeh; Rinder, C. A.; Simpson, Marcia V.; Smith, Paul

doi:10.1111/j.1749-6632.1984.tb13883.x

Cited by 4 publications

(3 citation statements)

References 3 publications

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“…the metal beam was rough and showed considerable relief while the other, which was seen only rarely, appeared smooth (24). Since it has been demonstrated that most of the mass of the enzyme protruding from the membrane is located on the cytoplasmic side (e.g., reference 30), we attribute the rough surface of the sheets to the cytoplasmic side of the plasma membrane.…”

Section: Dimeric Sheets Of Na K-atpasementioning

confidence: 78%

“…This was a region ~,6.0 nm in depth, abutting one side of the membrane. Since the majority of the molecular mass is located on the cytoplasmic side of the membrane (30), and little structure can be visualized on the exoplasmic side by freeze-drying (24), we attribute the structure we observe to the cytoplasmic side of the membrane only. Fig.…”

Section: The Three-dimensional Reconstructionmentioning

confidence: 82%

“…The limitation of recoverable information to one side of the membrane has been a common finding in three-dimensional studies of negatively stained specimens of membrane proteins, for example in the bladder membrane (3,22) and more recently in the reconstruction of the Ca-ATPase (38). In the case of the Na,K-ATPase an explanation may be found in resuits from freeze-dried and shadowed specimens of ATPase sheets (24) where the exoplasmic surface of the membrane, on the rare occasions it was exposed, was found to show little structure. It is possible that the extended sugar chains attached to the 13 subunit may collapse during specimen preparation forming a poorly organized meshwork that masks the regular structure of the exoplasmic protein component of the sheet.…”

Section: Discussionmentioning

confidence: 99%

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The three-dimensional structure of the Na,K-ATPase from electron microscopy.

Mohraz¹,

Simpson²,

Smith³

1987

The Journal of Cell Biology

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Abstract. The structure of Na,K-ATPase has been studied by electron microscopy and image reconstruction. A three-dimensional structure of this enzyme has been obtained to an overall resolution of 2.5 nm using data from specimens of negatively stained dimer sheets tilted through a range of angles +60 °.The reconstruction shows a complex mass distribution consisting of ribbons of paired molecules extending ,',,6.0 nm from the cytoplasmic side of the membrane. The molecular envelope consists of a massive "body" with "lobe" and "arm" structures projecting from it. The body has a columnar shape and is tilted with respect to the plane of the membrane. The region of interaction responsible for dimer formation is located between two bodies and is clearly visible in the reconstruction. It has been identified as a segment in the amino-terminal portion of the ct subunit. The arms that interconnect the ribbons are located close to the membrane and are most probably formed by the 13 subunits. NA,K-ATPaSe constitutes the ion pump that couples the hydrolysis of ATP to the transmembrane transport of sodium and potassium ions. It is primarily responsible for the establishment and maintenance of the gradients of Na + and K ÷ across the plasma membrane of eukaryotic cells. The transmembrane potential produced by this process is used by the cell to power other transport processes. The enzyme is built from two polypeptide chains (7,14,16,20), of which ¢t (Mr 110,000) is the catalytic subunit and 13 (mr 50,000-60,000) is a glycoprotein whose function has yet to be determined.The availability of purified specimens of Na,K-ATPase has allowed extensive studies of its various properties (for reviews see references 5, 10, 17, 32). The recent progress in structural studies of the enzyme has been particularly impressive. The amino acid sequence of ¢t (18, 34) and 13 (19,29,33) have been determined from various tissues. Furthermore, the discovery of crystallization schemes that produced regular arrays of the enzyme in membrane fragments (35) initiated structural studies by electron microscopy and image processing. Structural analyses of the enzyme in projection have been reported (11,23,42) as well as preliminary data on its three-dimensional structure (13,27,31). In this laboratory we have tentatively mapped the domains in the projection image of the enzyme that correspond to its two subunits (23, 25), and have found reliable crystallization methods to produce regular sheets of the enzyme in ap2 lattice (26).In this paper we present a three-dimensional structural study of Na,K-ATPase to an overall resolution of 2.5 nm. The reconstruction shows the structure of the enzyme on the cgtoplasmic side of the membrane, and reveals that it consists of tilted columns of mass interconnected by a complex set of bridges. These bridges can be identified with the bonds responsible for dimer formation and the subsequent ribbon-ribbon interaction. A segment of the amino-terminal of the ct subunit can be located within the structure and a tentative identific...

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Section: Dimeric Sheets Of Na K-atpasementioning

confidence: 78%

Section: The Three-dimensional Reconstructionmentioning

confidence: 82%