Soluble and active renal Na,K-ATPase with maximum protein molecular mass 170,000 ± 9,000 daltons; formation of larger units by secondary aggregation

Brotherus, Jaakko; Møller, Jesper; Jørgensen, Peter Leth

doi:10.1016/s0006-291x(81)80075-7

Cited by 128 publications

(74 citation statements)

References 18 publications

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“…That the aggregates proved to be more trypsin-sensitive than unaggregated material in the same samples also implied that denaturation preceded aggregation and that not all of the ␣ was equally denatured at the time point used. Aggregation of detergent-solubilized Na,K-ATPase units has also been reported (31,54), but is unlikely to be related to the events observed here. The formation of anomalously-migrating ␣ subunit forms after heating Na,K-ATPase in SDS sample buffer (55) is also not related to the observations reported here.…”

Section: Figsupporting

confidence: 60%

Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ Subunit Association with the C-terminal Domain

Donnet

Arystarkhova

Sweadner

2001

Journal of Biological Chemistry

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Thermal denaturation can help elucidate protein domain substructure. We previously showed that the Na,K-ATPase partially unfolded when heated to 55°C (Arystarkhova, E., Gibbons, D. L., and Sweadner, K. J. (1995) J. Biol. Chem. 270, 8785-8796). The ␤ subunit unfolded without leaving the membrane, but three transmembrane spans (M8-M10) and the C terminus of the ␣ subunit were extruded, while the rest of ␣ retained its normal topology with respect to the lipid bilayer. Here we investigated thermal denaturation further, with several salient results. First, trypsin sensitivity at both surfaces of ␣ was increased, but not sensitivity to V8 protease, suggesting that the cytoplasmic domains and extruded domain were less tightly packed but still retained secondary structure. Second, thermal denaturation was accompanied by SDS-resistant aggregation of ␣ subunits as dimers, trimers, and tetramers without ␤ or ␥ subunits. This implies specific ␣-␣ contact. Third, the ␥ subunit, like the C-terminal spans of ␣, was selectively lost from the membrane. This suggests its association with M8-M10 rather than the more firmly anchored transmembrane spans. The picture that emerges is of a Na,K-ATPase complex of ␣, ␤, and ␥ subunits in which ␣ can associate in assemblies as large as tetramers via its cytoplasmic domain, while ␤ and ␥ subunits associate with ␣ primarily in its C-terminal portion, which has a unique structure and thermal instability.

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Section: Figsupporting

confidence: 60%

Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ Subunit Association with the C-terminal Domain

Donnet

Arystarkhova

Sweadner

2001

Journal of Biological Chemistry

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“…The assay mixture was incubated at 37°C for 30 min, and the phosphate release was determined as reported by Brotherus et al (24). The Na,K-ATPase activity was the difference between the ATP hydrolysis measured in the presence and absence of 83 M ouabain.…”

Section: Methodsmentioning

confidence: 99%

Site-directed Chemical Labeling of Extracellular Loops in a Membrane Protein

Kaplan

2000

Journal of Biological Chemistry

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We have mapped the membrane topology of the renal Na,K-ATPase ␣-subunit by using a combination of introduced cysteine mutants and surface labeling with a membrane impermeable Cys-directed reagent, N-biotinylaminoethyl methanethiosulfonate. To begin our investigation, two cysteine residues (Cys 911 and Cys 964 ) in the wild-type ␣-subunit were substituted to create a background mutant devoid of exposed cysteines (Lutsenko, S., Daoud, S., and Kaplan, J. H. (1997) J. Biol. Chem. 272, 5249 -5255). Into this background construct were then introduced single cysteines in each of the five putative extracellular loops (P118C, T309C, L793C, L876C, and M973C) and the resulting ␣-subunit mutants were co-expressed with the ␤-subunit in baculovirusinfected insect cells. All of our expressed Na,K-ATPase mutants were functionally active. Their ATPase, phosphorylation, and ouabain binding activities were measured, and the turnover of the phosphoenzyme intermediate was close to the wild-type enzyme, suggesting that they are folded properly in the infected cells. Incubation of the insect cells with the cysteine-selective reagent revealed essentially no labeling of the ␣-subunit of the background construct and labeling of all five mutants with single cysteine residues in putative extracellular loops. Two additional mutants, V969C and L976C, were created to further define the M9M10 loop. The lack of labeling for these two mutants showed that although Met 973 is apparently exposed, Val 969 and Leu 976 are not, demonstrating that this method may also be utilized to define membrane aqueous boundaries of membrane proteins. Our labeling studies are consistent with a specific 10-transmembrane segment model of the Na,KATPase ␣-subunit. This strategy utilized only functional Na,K-ATPase mutants to establish the membrane topology of the entire ␣-subunit, in contrast to most previously applied methods. Na,K-ATPase (sodium pump) is an integral membrane protein that is present in most animal cells. The enzyme consists of two subunits, a large catalytic ␣-subunit (about 110 kDa) and a glycosylated ␤-subunit (ϳ55 kDa); both subunits are required for enzymatic activity (1-3). Na,K-ATPase utilizes the energy derived from ATP hydrolysis to transport Na ϩ and K ϩ ions across plasma membranes against their electrochemical gradients and is a member of the P 2 -ATPase family (4). The ion gradients generated by the sodium pump are important for regulating a variety of physiological functions such as cell excitability, contractility, and secondary active transport. Several isoforms of the ␣-and ␤-subunits have been cloned from various species, and the primary sequences have been described (5). The secondary structural information on the protein, on the other hand, remains controversial despite extensive investigation; such information is essential for understanding structure/function relationships of the Na,K-ATPase. Based on hydropathy analysis (6), protease accessibility (7, 8), and immunochemical studies (9, 10), the amino-terminal third of the ␣-subun...

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“…Rabbit kidney Na,K-ATPase, when solubilized with C 12 E 8 , and purified by fractionated centrifugation, showed that the solubilized form of the enzyme consists predominantly (80-85%) of associations of the (aß) type, with molecular mass between 140 and 170 kDa (7,(20)(21)(22)28). However, when other isolation methods are used, such as gel filtration, a relatively stable association of subunit (aß) 2 types and/or superior oligomers with molecular mass of 257-380 kDa was observed.…”

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

“…However, when other isolation methods are used, such as gel filtration, a relatively stable association of subunit (aß) 2 types and/or superior oligomers with molecular mass of 257-380 kDa was observed. It is important to note that these associations are necessary for the enzyme to have a catalytic activity and are related to the incubation time with the detergent and to detergent concentration (10,(20)(21)(22)24).…”

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

Santos

Lamas

Ciancaglini

2002

Braz J Med Biol Res

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SDS, C 12 E 8 , CHAPS or CHAPSO or a combination of two of these detergents is generally used for the solubilization of Na,K-ATPase and other ATPases. Our method using only C 12 E 8 has the advantage of considerable reduction of the time for enzyme purification, with rapid solubilization and purification in a single chromatographic step. Na,KATPase-rich membrane fragments of rabbit kidney outer medulla were obtained without adding SDS. Optimum conditions for solubilization were obtained at 4ºC after rapid mixing of 1 mg of membrane Na,K-ATPase with 1 mg of C 12 E 8 /ml, yielding 98% recovery of the activity. The solubilized enzyme was purified by gel filtration on a Sepharose 6B column at 4ºC. Non-denaturing PAGE revealed a single protein band with phosphomonohydrolase activity. The molecular mass of the purified enzyme estimated by gel filtration chromatography was 320 kDa. The optimum apparent pH obtained for the purified enzyme was 7.5 for both PNPP and ATP. The dependence of ATPase activity on ATP concentration showed high (K 0.5 = 4.0 µM) and low (K 0.5 = 1.4 mM) affinity sites for ATP, with negative cooperativity. Ouabain (5 mM), oligomycin (1 µg/ml) and sodium vanadate (3 µM) inhibited the ATPase activity of C 12 E 8 -solubilized and purified Na,KATPase by 99, 81 and 98.5%, respectively. We have shown that Na,KATPase solubilized only with C 12 E 8 can be purified and retains its activity. The activity is consistent with the form of (aß) 2 association.

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Soluble and active renal Na,K-ATPase with maximum protein molecular mass 170,000 ± 9,000 daltons; formation of larger units by secondary aggregation

Cited by 128 publications

References 18 publications

Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ Subunit Association with the C-terminal Domain

Thermal Denaturation of the Na,K-ATPase Provides Evidence for α-α Oligomeric Interaction and γ Subunit Association with the C-terminal Domain

Site-directed Chemical Labeling of Extracellular Loops in a Membrane Protein

Solubilization of Na,K-ATPase from rabbit kidney outer medulla using only C12E8

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