Molecular weights of αβ-protomeric and oligomeric units of soluble (Na+, K+)-ATPase determined by low-angle laser light scattering after high-performance gel chromatography

Hayashi, Yutaro; Takagi, Toshio; Maezawa, Shigenori; Matsui, Hideo

doi:10.1016/0167-4838(83)90291-1

Cited by 61 publications

(23 citation statements)

References 38 publications

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“…It is clearly involved in the transport cycle, as the reductive loss of its intramolecular Cys-Cys bonds results in the loss of the ability to occlude K ϩ ions and the associated loss of Na-K-ATPase activity (29), and it undergoes conformational transitions in concert with the ␣-subunit during the reaction cycle (8,25). Studies in Madin-Darby canine kidney (MDCK) cells (17) confirmed the 1:1 ␤-to-␣ ratio previously reported for purified enzyme (7). Recent studies suggest that, in addition to enabling transport and stabilization of the Na-KATPase complex in the PM, ␤ 1 may be involved in the structural organization of epithelial cell monolayers.…”

supporting

confidence: 83%

“…Similar results were obtained in MDCK/␤ 1 myc cells and in HEK-293 cells overexpressing ␤ 1 flag (data not shown). If normal MDCK cells have an equimolar ratio of assembled ␣ 1 -and ␤ 1 -subunits (7,17,20), the densitometric analysis of ␤-subunits associated with ␣-subunit, represented in Fig. 5, A and B, suggests that the induced MDCK/␤ 1 flag cells have approximately a 1:2 to 1:5 ratio of interacting ␣ 1 -to ␤ 1 -subunits, depending on the extent of ␤ 1 flag overexpression.…”

Section: Protein Expression and Membrane Distribution In Mdck/mentioning

confidence: 99%

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β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

Clifford¹,

Kaplan²

2008

American Journal of Physiology-Renal Physiology

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Clifford RJ, Kaplan JH. ␤-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells. Am J Physiol Renal Physiol 295: F1314 -F1323, 2008. First published August 13, 2008 doi:10.1152/ajprenal.90406.2008In eukaryotic cells, the apparent maintenance of 1:1 stoicheometry between the Na-K-ATPase ␣-and ␤-subunits led us to question whether this was alterable and thus if some form of regulation was involved. We have examined the consequences of overexpressing Na-K-ATPase ␤ 1-subunits using Madin-Darby canine kidney (MDCK) cells expressing flag-tagged ␤ 1-subunits (␤1flag) or Myc-tagged ␤1-subunits (␤1myc) under the control of a tetracycline-dependent promoter. The induction of ␤ 1flag subunit synthesis in MDCK cells, which increases ␤1-subunit expression at the plasma membrane by more than twofold, while maintaining stable ␣ 1 expression levels, revealed that all mature ␤ 1-subunits associate with ␣1-subunits, and no evidence of "free" ␤ 1-subunits was obtained. Consequently, the ratio of assembled ␤1-to ␣1-subunits is significantly increased when "extra" ␤-subunits are expressed. An increased ␤ 1/␣1 stoicheometry is also observed in cells treated with tunicamycin, suggesting that the protein-protein interactions involved in these complexes are not dependent on glycosylation. Confocal images of cocultured ␤ 1myc-expressing and ␤1flag-expressing MDCK cells show colocalization of ␤1myc and ␤1flag subunits at the lateral membranes of neighboring cells, suggesting the occurrence of intercellular interactions between the ␤-subunits. Immunoprecipitation using MDCK cells constitutively expressing ␤1myc and tetracycline-regulated ␤1flag subunits confirmed ␤-␤-subunit interactions. These results demonstrate that the equimolar ratio of assembled ␤1/

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supporting

confidence: 83%

Section: Protein Expression and Membrane Distribution In Mdck/mentioning

confidence: 99%

β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

Clifford¹,

Kaplan²

2008

American Journal of Physiology-Renal Physiology

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“…The ␣ 1 and ␤ 1 subunits of sodium pump found in MDCK cells (21) or enzyme purified from dog kidney (43,44) are expressed at an equal ratio and form into ␣/␤ heterodimers. To maintain equimolar ratios the cells must possess mechanisms that regulate subunit abundance.…”

Section: Polypeptide Coding Sequence Is Involved In the Regulation-mentioning

confidence: 99%

Regulation of Na,K-ATPase Subunit Abundance by Translational Repression

Clifford

Kaplan

2009

Journal of Biological Chemistry

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The Na,K-ATPase is an ␣␤ heterodimer responsible for maintaining fluid and electrolyte homeostasis in mammalian cells. We engineered Madin-Darby canine kidney cell lines expressing ␣ 1 FLAG, ␤ 1 FLAG, or ␤ 2 MYC subunits via a tetracycline-regulated promoter and a line expressing both stable ␤ 1 MYC and tetracycline-regulated ␤ 1 FLAG to examine regulatory mechanisms of sodium pump subunit expression. When overexpression of exogenous ␤ 1 FLAG increased total ␤ subunit levels by >200% without changes in ␣ subunit abundance, endogenous ␤ 1 subunit (␤ 1 E) abundance decreased. ␤ 1 E downregulation did not occur during ␤ 2 MYC overexpression, indicating isoform specificity of the repression mechanism. Measurements of RNA stability and content indicated that decreased ␤ subunit expression was not accompanied by any change in mRNA levels. In addition, the degradation rate of ␤ subunits was not altered by ␤ 1 FLAG overexpression. Cells stably expressing ␤ 1 MYC, when induced to express ␤ 1 FLAG subunits, showed reduced ␤ 1 MYC and ␤ 1 E subunit abundance, indicating that these effects occur via the coding sequences of the down-regulated polypeptides. In a similar way, Madin-Darby canine kidney cells overexpressing exogenous ␣ 1 FLAG subunits exhibited a reduction of endogenous ␣ 1 subunits (␣ 1 E) with no change in ␣ mRNA levels or ␤ subunits. The reduction in ␣ 1 E compensated for ␣ 1 FLAG subunit expression, resulting in unchanged total ␣ subunit abundance. Thus, regulation of ␣ subunit expression maintained its native level, whereas ␤ subunit was not as tightly regulated and its abundance could increase substantially over native levels. These effects also occurred in human embryonic kidney cells. These data are the first indication that cellular sodium pump subunit abundance is modulated by translational repression. This mechanism represents a novel, potentially important mechanism for regulation of Na,K-ATPase expression.

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“…Bearing in mind the data on the equimolarity of the cy-and ,f?-polypeptides in the enzyme molecule [4, one can conclude further that 2 a-subunits are present as well, half of them modified in such a way that they cannot be detected at NHz-terminal analysis; presumably they bear a blocked a-amino group.…”

Section: Resultsmentioning

confidence: 94%

“…Earlier studies on the maximal capacity of the purest and most active preparations to bind specific ligands revealed 3.5-4.0 nmof lig~d-bin~ng units per mg Lowry protein [ I,21 suggesting the molecular mass of the unit to be 3250 kDa which is roughly twice as great as that of the a,-protomer [3,4]. These data were, however, disregarded later in reports where the Na,K-ATPase protein concentration was assessed by amino acid analysis and an essentially lower value than that found in the Lowry assay was obtained [5-71. This observation led to the conclusion that each single &-protomer serves as a ligand-binding (and probably enzyme) unit [8].…”

Section: Introductionmentioning

confidence: 99%

Evidence for a diprotomeric structure of Na,K‐ATPase

Chetverin

1986

FEBS Letters

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Three methods were used to assess protein ~n~nt~tion in m~br~5bound Na,K-ATPase preparations: standard Lowry assay, KjeldahI nitrogen determination and amino acid analysis. While the first two methods showed excellent agreement, the third one always gave a lower value which varied drastically depending on the condition of sample treatment before amino acid analysis. This result reinforces the Lowry method in assessing the true concentration of Na,K-ATPase protein and suggests 250 kDa to be a true estimate of the molecular mass of the smallest ligand-binding unit of the enzyme. The cyanate method reveals two NH,-terminal residues of the /J-subunit (NH,-Ala) and one such residue of the a-subunit (NH,-Gly) per ligand-binding unit. From the data on equimolarity of the a-and /I-subunits in Na,K-ATPase this suggests that the enzyme molecule is composed of two afl-protomers, one possessing a modified (presumably an Nblocked) a-subunit.(Na+ + K+)-ATPase Enzyme structure Subunit modt$cation Protein assay End-group analysis

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Molecular weights of αβ-protomeric and oligomeric units of soluble (Na+, K+)-ATPase determined by low-angle laser light scattering after high-performance gel chromatography

Cited by 61 publications

References 38 publications

β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

β-Subunit overexpression alters the stoicheometry of assembled Na-K-ATPase subunits in MDCK cells

Regulation of Na,K-ATPase Subunit Abundance by Translational Repression

Evidence for a diprotomeric structure of Na,K‐ATPase

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