Three‐dimensional structure of renal Na,K‐ATPase determined by electron microscopy of membrane crystals

Herbert, Hans; Skriver, Elisabeth; Maunsbach, Arvid B.

doi:10.1016/0014-5793(85)81238-2

Cited by 48 publications

(25 citation statements)

References 13 publications

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“…4). Their asymmetric localization within the membrane is consistent with the asymmetric location of the stain-excluding region interpreted as the lipid bilayer in three-dimensional reconstructions (8,15).…”

Section: Resultssupporting

confidence: 73%

“…In the dimeric crystals the two protomers have been observed in projection images to be similar (4) or dissimilar (2). Also three-dimensional reconstructions of the Na,K-ATPase protein in dimeric crystals have shown unit cells with either two identical protomers (8,18,20) or two differently shaped protomers in each unit cell (6). Recent cryo-electron microscopy of unfixed and unstained two-dimensional cryostals of Na,KATPase in frozen-hydrated preparations have confirmed that the projection structure of the enzyme protein in the crystalline arrays are either dimeric (16,17) or monomeric (16).…”

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confidence: 99%

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Cryo-electron microscope analysis of frozen-hydrated crystals of Na, K-ATPase.

Maunsbach

Hebert²,

Kavéus³

1992

Acta Histochem. Cytochem

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Two-dimensional membrane crystals were induced in purified preparations of membrane-bound Na,K-ATPase. The crystals were analyzed by cryo-electron microscopy in frozen-hydrated preparations using minimal dose conditions and elastic brightfield. The vanadate-induced crystals showed predominantly p1 or p21 symmetries and image analysis using correlation averaging demonstrated that the protomers in dimeric crystals were either similar or different in the projected structure. The observations suggest that the Na, K-ATPase protomes are gradually reorganized within the membrane crystals during the assembly process.

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Section: Resultssupporting

confidence: 73%

mentioning

confidence: 99%

Cryo-electron microscope analysis of frozen-hydrated crystals of Na, K-ATPase.

Maunsbach

Hebert²,

Kavéus³

1992

Acta Histochem. Cytochem

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“…The Na+,K+-ATPase protein is composed of an a and a (3 subunit and is probably present in the membrane as an (a,3)2 diprotomer. Taking the measured lipid/protein ratio of 350 mol of lipid per mol of (a,8)2 dimer, an area per lipid molecule of 0.6 nm2, and the extramembranous dimensions of the (a3)2 Na+,K+-ATPase dimer of 8 x 6 nm (22) yields values of konol = 0.5 x 106, 1.9 x 106, and 4.4 x 106 s51 at 40C, 150C, and 250C, respectively. These values for the collision rate constant have been calculated by assuming a value of T, = 1 ps (20).…”

Section: Resultsmentioning

confidence: 99%

“…This lattice model is admittedly an oversimplification for protein diffusion, but it should give the correct order of magnitude with appropriate choice of the characteristic length, A. Values for the translational diffusion coefficient of the Na+,K+-ATPase obtained for a value of A 6 nm (22) are then 0.8, 2.9, and 6.6 gm2-s-1 at 40C, 15TC, and 25°(, respectively. These values were calculated by assuming that a: 1, which is consistent with the relatively high degree of labeling of the protein and the surface location of many of the labeled groups (see ref.…”

Section: Resultsmentioning

confidence: 99%

Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

Esmann

Marsh

1992

Proc. Natl. Acad. Sci. U.S.A.

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Diffusion-controlled Heisenberg spin exchange between spin-labeled Na+,K(+)-ATPase [ATP phosphohydrolase (Na+/K(+)-transporting), EC 3.6.1.37] proteins has been studied by saturation transfer ESR spectroscopy in reconstituted membranes. Na+,K(+)-ATPase from the salt gland of Squalus acanthias was solubilized in a polyoxyethylene ether detergent, octa(ethylene glycol) dodecyl monoether. Part of the solubilized enzyme was covalently spin-labeled with a nitroxide derivative of indanedione and recombined with various proportions of the unlabeled enzyme while the native lipid/protein ratio was maintained. Purified membranes were then reconstituted from the various samples by precipitation with divalent ions. The reciprocal integrated intensities of the saturation transfer ESR spectra were found to increase linearly with the fraction of protein that was spin-labeled, and the gradient of the concentration dependence increased with increasing temperature over the range 4 degrees-25 degrees C. Comparison with theoretical analyses of the effects of weak Heisenberg spin exchange [Marsh, D. & Horváth, L. I. (1992) J. Magn. Reson. 97, 13-26] suggests that the effects on the saturation transfer ESR intensity are attributable to short-range diffusional collisions between the spin-labeled protein molecules. The effective value of the local translational diffusion coefficient is 1.8-2.9 microns2.s-1 at 15 degrees C, depending on the diffusion model used, which is much larger than the values obtained for the long-range diffusion coefficient in cells by photobleaching techniques. The temperature dependence of the translational diffusion is larger than expected but correlates with the anomalous temperature dependence of the rotational diffusion observed in the same system.

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“…Occupation of the lowaffinity ATP binding site in this model does not affect the affinity and the capacity of the high-affinity ATP binding site; but fixing ATP at either binding site blocks the catalytic activity and the turnover of both subunits. Evidence for a dimeric nature of (Na' + K+)-ATPase has been obtained from radiation inactivation studies [59, 601, molecular mass determinations [61, 621, cross-linking experiments [63, 641 and the three-dimensional analysis of electron microscopic photographs [65,661. We conclude therefore that the experimental results obtained with C O ( N H~)~A T P are consistent with a model of interacting catalytic subunits in the mechanism of (Na' + K+)-ATPase.…”

Section: Binding Of (3h]ouubuin To the C~( N H~)~a T P -I N A C T I Vmentioning

confidence: 99%

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

1987

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The MgATP complex analogue cobalt-tetrammine-ATP [CO(NH~)~ATP] inactivates (Na' + K ')-ATPase at Despite the inactivation of (Na' + K+)-ATPase by C O ( N H~)~A T P from the low-affinity ATP binding site, there is no change in the capacity of the high-affinity ATP binding site (Kd = 0.9 pM) nor of its capability to phosphorylate the enzyme Na+-dependently. Since (Na' + K+)-ATPase is phosphorylated Na+-dependently from the high-affinity ATP binding site although the catalytic cycle is arrested in the E2 conformational state by specific modification of the low-affinity ATP binding site, it is concluded that both ATP binding sites coexist at the same time in the working sodium pump. This demonstration of interacting catalytic subunits in the El and E2 conformational states excludes the proposal that a single catalytic subunit catalyzes (Na' + K +)-transport.Active transport of sodium and potassium through cellular membranes catalyzed by (Na' + K+)-ATPase is accompanied by conformational changes of the enzyme protein [I -41. High-and low-affinity ATP binding sites and a nonhyperbolic ATP saturation curve of the catalytic activity seem to be an expression this [5 -1 I]. While binding of ATP to the high-affinity ATP binding site appears to be essential for the Correspondence to G.

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Three‐dimensional structure of renal Na,K‐ATPase determined by electron microscopy of membrane crystals

Cited by 48 publications

References 13 publications

Cryo-electron microscope analysis of frozen-hydrated crystals of Na, K-ATPase.

Cryo-electron microscope analysis of frozen-hydrated crystals of Na, K-ATPase.

Local translational diffusion rates of membranous Na+,K(+)-ATPase measured by saturation transfer ESR spectroscopy.

Demonstration of cooperating alpha subunits in working (Na+ + K+)-ATPase by the use of the MgATP complex analogue cobalt tetrammine ATP

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