Soluble and enzymatically stable (Na++K+)-ATPase from mammalian kidney consisting predominantly of protomer αβ-units

Brotherus, Jaakko; Jacobsen, Lene; Jørgensen, Peter Leth

doi:10.1016/0005-2736(83)90021-4

Cited by 124 publications

(73 citation statements)

References 36 publications

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“…Under these conditions, the specific ATPase activity of the solubilized Na,K-ATPase was in the range of 600-700 U/mg, which is similar to the activity of the membrane enzyme, as reported by Cornelius (7) for the enzyme obtained from rabbit kidney. However, activities 20-30-fold higher have been reported both for membrane-bound and solubilized enzyme (8,10,28). In our study the initial activity for the membrane-bound enzyme was lower than reported by others but a purification factor of about 6-10-fold, which is typical for enzyme isolated from membrane of specialized tissue, suggests a selective extraction of Na,K-ATPase.…”

Section: Nak-atpase Solubilized Using Only C 12 Econtrasting

confidence: 62%

“…The development of a rational approach to the solubilization of membrane proteins always requires a review of the literature to determine the detergent type and the conditions that are generally used for the solubilization of the protein of interest (1,(3)(4)(5)(6)(7)(8)10). All assays were carried at 37ºC as described in Methods.…”

Section: Discussionmentioning

confidence: 99%

“…Our method using only C 12 E 8 is based on the methodology described by Santos and Ciancaglini (5) and others who used it combined with other detergents (7,10). The advantage of this method is the considerable reduction in the time needed to obtain the purified enzyme, which is solubilized instantaneously and purified in a single chromatographic step and has a smaller denaturing effect on the solubilized enzyme.…”

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

“…A detergent that has been used with great success in Na,K-ATPase solubilization is C 12 E 8 (4,5,7,10,11). Different results showed that Na,K-ATPase previously treated with SDS can be solubilized with C 12 E 8 in an active form where most of the kinetic and conformational properties of the enzyme are preserved after solubilization (4,7,11).…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Nak-atpase Solubilized Using Only C 12 Econtrasting

confidence: 62%

Section: Discussionmentioning

confidence: 99%

Section: Nak-atpase Solubilized Using Only C 12 Ementioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

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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“…Na,K-ATPase treatment with C 12 E 8 has been suggested to ensure that the enzyme is a single functional protomer (i.e. a single α/β complex) [30,31]. We also reexamined eosin and ATP competition for Na,K-ATPase on C 12 E 8 -treated enzyme, but even in this case eosin appeared to be a mixed-type rather than a competitive inhibitor (data not shown).…”

Section: Resultsmentioning

confidence: 95%

Kinetic characterization of Na,K-ATPase inhibition by Eosin

Ogan

Reifenberger

Milanick

et al. 2007

Blood Cells, Molecules, and Diseases

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Eosin is a probe for the Na pump nucleotide site. In contrast to previous studies examining eosin effects on Na only ATPase, we examined Na,K ATPase and K activated pNPPase activity in red blood cell membranes and purified renal Na,K ATPase. At saturating ATP (3mM) the eosin IC 50 for Na pump inhibition was 19uM. Increasing ATP concentrations (0.2 -2.5 mM) did not overcome eosin-induced inhibition thus eosin is a mixed-type inhibitor of ATPase activity. To test if eosin can bind to the high affinity ATP site, purified Na,K ATPase was labeled with 20 uM FITC. With increasing eosin concentrations (0.1 uM -10 uM) the incorporation of FITC into the ATP site significantly decreases suggesting that eosin prevents FITC reaction at the high affinity ATP site. Eosin was a more potent inhibitor of K activated phosphatase activity than of Na,K ATPase activity. At 5mM pNPP the eosin IC50 for Na pump inhibition was 3.8 ± 0.23 uM. Increasing pNPP concentrations (0.45 -14.5 mM) did not overcome eosin-induced inhibition thus eosin is a mixedtype inhibitor of pNPPase activity. These results can be fit by a model in which eosin and ATP bind only to the nucleotide site; in some pump conformations, this site is rigid and the binding is mutually exclusive and in other conformations, the site is flexible and able to accommodate both eosin and ATP (or pNPP). Interestingly, eosin inhibition of pNPPase became competitive after the addition of C 12 E 8 (0.1%) but the inhibition of ATPase remained mixed.

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Mechanismen des biologischen Ionentransports – Carrier, Kanäle und Pumpen in künstlichen Lipidmembranen

Läuger

1985

Angew. Chem.

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Angew. Chem. 97 (1985) 939-959 0 V C H Verlagsgesellschafr mbH. 0-6940 Weinheim. 1985 0044-8249/85/1 I 11-0939 S 02.50/0 Angew. Chem. 97 (1985) 939-959 U. Wilmsen, C. Methfessel, W. Hanke, G. Boheim in G. Spach (Hrsg.): Physical Chemisfiy of Transmembrane Ion Mofions. Elsevier, Amsterdam 1983. W. Hanke, C. Methfessel, U. Wilmsen, G. Boheim, Eioelecrrochem. Bioenerg. 12 (1984) 329. R. Coronado, R. Latorre, Eiophys. J. 43 (1983) 23 I . B. A. Suarez-Isla, K. Wan, J. Lindstrom, M. Montal, Biochemistry 22 (1983) 2319. T. Schuerholz, H. Schindler, FEES Lerr. I52 (1983) 187. A. D. Bangham (Hrsg.): Lipomme Lerrers. Academic Press, New York 1983. C . G. Knight (Hrsg.): Liposomes: From Physical Srrucrure 10 Therapeuric Applicafions. Elsevier, Amsterdam 198 1. H.-J. Apell, M. M. Marcus, B. M. Anner, H. Oetliker. P. Lauger, J . Membr. Eiol. 85 (1985) 49. J. H. Fendler: Membrane Mimeric Chemisrry.

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Soluble and enzymatically stable (Na++K+)-ATPase from mammalian kidney consisting predominantly of protomer αβ-units

Cited by 124 publications

References 36 publications

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

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

Kinetic characterization of Na,K-ATPase inhibition by Eosin

Mechanismen des biologischen Ionentransports – Carrier, Kanäle und Pumpen in künstlichen Lipidmembranen

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