Large-scale preparation of sodium-potassium ATPase from kidney outer medulla

Klodos, Irena; Esmann, Mikael; Post, Robert L.

doi:10.1046/j.1523-1755.2002.00654.x

Cited by 90 publications

(68 citation statements)

References 17 publications

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“…Purified pig kidney Na + , K + -ATPase was prepared as previously described (16). The specific activity was about 1,800 μmol/mg protein per hour at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Structures and characterization of digoxin- and bufalin-bound Na ⁺ ,K ⁺ -ATPase compared with the ouabain-bound complex

Laursen

Gregersen

Yatime

et al. 2015

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

201

269

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Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na + ,K + -ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five-or six-membered lactone. These functionalities have specific influence on the binding properties. We report crystal structures of the Na + ,K + -ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare their characteristics and binding kinetics with the previously described E2P-ouabain complex to derive specific details and the general mechanism of CTS binding and inhibition. CTSs block the extracellular cation exchange pathway, and cation-binding sites I and II are differently occupied: A single Mg 2+ is bound in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K + in the E2P-bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type of the lactone substituent determine the arrangement of αM4 and hypothesize that winding/unwinding of αM4 represents a trigger for high-affinity CTS binding. We find that the level of glycosylation affects the depth of CTS binding and that the steroid core substituents fine tune the configuration of transmembrane helices αM1-2.Na/K-ATPase | phosphoenzyme | inhibitor | cardiac glycosides | structure C ardiotonic steroids (CTSs) induce diverse physiological effects on, for example, heart muscle and blood pressure regulation, but the underlying mechanisms remain unknown, despite a long history of therapeutic applications and model studies. It is widely recognized that they target Na + ,K + -ATPase, and a direct consequence of their binding is an inhibition of the enzyme. Their positive inotropic effect in cardiomyocytes has been related to coupling between Na + ,K + -ATPase and Na + /Ca 2+ -exchanger through the intracellular Na + concentration, whereas numerous other outcomes observed on the cellular level have led to hypotheses of the existence of signaling cascade mechanisms with Na + ,K + -ATPase acting as a receptor. The minimal functional unit of the enzyme is an αβ-complex, and because there exist four α-and three β-isoforms of the Na + ,K + -ATPase, the variations in the heterodimer composition and a vast number of CTSs differing in apparent isoform specificities (1) add to the complexity and multiplicity of reported physiological responses.The conserved structural core shared by all CTSs includes a cis-trans-cis ring-fused steroid core with two methyl substituents at steroid positions C10 β and C13 β , two hydroxyl groups (OH3 β and OH14 β ), and an unsaturated lactone ring at the C17 β position, among which the lactone and OH14 β are critical for binding to Na + ,K + -ATPase (2, 3). The type of lactone at the C...

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“…Purified pig kidney Na + , K + -ATPase was prepared as previously described (16). The specific activity was about 1,800 μmol/mg protein per hour at 37°C.…”

Section: Methodsmentioning

confidence: 99%

Structures and characterization of digoxin- and bufalin-bound Na ⁺ ,K ⁺ -ATPase compared with the ouabain-bound complex

Laursen

Gregersen

Yatime

et al. 2015

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

201

269

View full text Add to dashboard Cite

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“…13), is separated from the sedimented purified enzyme in the course of purification (Table I). We conclude that the properties of the membrane-bound Na ϩ /K ϩ -ATPase preparations that are routinely purified by the classical SDS treatment (21), or its modifications (54,55) are not distorted by SDS, provided that the preparations are appropriately diluted and washed to be freed of the solubilized and partially unfolded enzyme. In the absence of experimental data demonstrating the production of the unfolded enzyme during purification by causes other than SDS, we also conclude that the findings on the purified enzyme showing its ligand-induced negative cooperativity of binding and partial site reactivity (5,6,41,42) are not preparationinduced artifacts.…”

Section: Table Imentioning

confidence: 99%

Interaction of SDS with Na+/K+-ATPase

Ivanov

Gable

Askari

2004

Journal of Biological Chemistry

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Because nearly all structure/function studies on Na ؉ / K ؉ -ATPase have been done on enzymes prepared in the presence of SDS, we have studied previously unrecognized consequences of SDS interaction with the enzyme. When the purified membrane-bound kidney enzyme was solubilized with SDS or TDS concentrations just sufficient to cause complete solubilization, but not at concentrations severalfold higher, the enzyme retained quaternary structure, exhibiting ␣,␣-, ␣,␤-, ␤,␤-, and ␣,␥-associations as detected by chemical cross-linking. The presence of solubilized oligomers was confirmed by sucrose density gradient centrifugation. This solubilized enzyme had no ATPase activity and was not phosphorylated by ATP, but it retained the ability to occlude Rb ؉ and Na ؉ . This, and comparison of cross-linking patterns obtained with different reagents, suggested that the transmembrane domains of the enzyme are more resistant to SDSinduced unfolding than its other domains. These findings (a) indicate that the partially unfolded oligomer(s) retaining partial function is the intermediate in the SDS-induced denaturation of the native membrane enzyme having the minimum oligomeric structure of (␣,␤,␥) 2 and (b) suggest potential functions for Na ؉ /K ؉ -ATPase with intrinsically unfolded domains. Mixtures of solubilized/ partially unfolded enzyme and membrane-bound enzyme exhibited cross-linking patterns and Na ؉ occlusion capacities different from those of either enzyme species, suggesting that the two interact. Formation of the partially unfolded enzyme during standard purification procedure for the preparation of the membrane-bound enzyme was shown, indicating that it is necessary to ensure the separation of the partially unfolded enzyme from the membrane-bound enzyme to avoid the distortion of the properties of the latter.

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“…Preparation of Rat Heart Sarcolemma Microsomes-Sarcolemma microsomes were prepared as described previously for preparation of kidney plasma membranes (43). Briefly, left ventricular rat cardiac tissue was homogenized in ISE buffer (25 mM imidazole, pH 7.3, 1 mM EDTA, and 250 mM sucrose).…”

Section: Expression Of Fxyd1 In E Coli Purification Of Fxyd1 and Rmentioning

confidence: 99%

Stabilization of the α2 Isoform of Na,K-ATPase by Mutations in a Phospholipid Binding Pocket

Kapri-Pardes

Katz

Haviv

et al. 2011

Journal of Biological Chemistry

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Background:The ␣2 isoform of Na,K-ATPase is unstable compared with ␣1 and ␣3. Results: Mutations in TM8 -10 strongly stabilize ␣2. A novel phospholipid antagonist selectively inactivates ␣2, and mutations in TM8 -10 protect against inactivation. Conclusion: A phosphatidylserine binding pocket within TM8 -10 has been identified. Significance: Mechanistic insights into ␣2 instability and a possible physiological role have been obtained.

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Large-scale preparation of sodium-potassium ATPase from kidney outer medulla

Abstract: There is a significant saving of labor to obtain a product with a specific activity similar to that commonly obtained. The microsomal fraction may be useful for preparing other membrane proteins from the outer medulla.

Cited by 90 publications

References 17 publications

Structures and characterization of digoxin- and bufalin-bound Na ⁺ ,K ⁺ -ATPase compared with the ouabain-bound complex

Structures and characterization of digoxin- and bufalin-bound Na ⁺ ,K ⁺ -ATPase compared with the ouabain-bound complex

Interaction of SDS with Na+/K+-ATPase

Stabilization of the α2 Isoform of Na,K-ATPase by Mutations in a Phospholipid Binding Pocket

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Large-scale preparation of sodium-potassium ATPase from kidney outer medulla

Abstract: There is a significant saving of labor to obtain a product with a specific activity similar to that commonly obtained. The microsomal fraction may be useful for preparing other membrane proteins from the outer medulla.

Cited by 90 publications

References 17 publications

Structures and characterization of digoxin- and bufalin-bound Na + ,K + -ATPase compared with the ouabain-bound complex

Structures and characterization of digoxin- and bufalin-bound Na + ,K + -ATPase compared with the ouabain-bound complex

Interaction of SDS with Na+/K+-ATPase

Stabilization of the α2 Isoform of Na,K-ATPase by Mutations in a Phospholipid Binding Pocket

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Structures and characterization of digoxin- and bufalin-bound Na ⁺ ,K ⁺ -ATPase compared with the ouabain-bound complex

Structures and characterization of digoxin- and bufalin-bound Na ⁺ ,K ⁺ -ATPase compared with the ouabain-bound complex