Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1996 Jan;70(1):182–193. doi: 10.1016/S0006-3495(96)79562-0

The carbohydrate moieties of the beta-subunit of Na+, K(+)-ATPase: their lateral motions and proximity to the cardiac glycoside site.

E Amler 1, A Abbott 1, H Malak 1, J Lakowicz 1, W J Ball Jr 1
PMCID: PMC1224919  PMID: 8770197

Abstract

The beta-subunit associated with the catalytic (alpha) subunit of the mammalian Na+, K(+) -ATPase is a transmembrane glycoprotein with three extracellularly located N-glycosylation sites. Although beta appears to be essential for a functional enzyme, the role of beta and its sugars remains unknown. In these studies, steady-state and dynamic fluorescence measurements of the fluorophore lucifer yellow (LY) covalently linked to the carbohydrate chains of beta have demonstrated that the bound probes are highly solvent exposed but restricted in their diffusional motions. Furthermore, the probes' environments on beta were not altered by Na+ or K+ or ouabain-induced enzyme conformational changes, but both divalent cation and oligomycin addition evoked modest changes in LY fluorescence. Frequency domain measurements reflecting the Förster fluorescence energy transfer (FET) occurring between anthroylouabain (AO) bound to the cardiac glycoside receptor site on alpha and the carbohydrate-linked LY demonstrated their close proximity (18 A). Additional FET determinations made between LY as donor and erythrosin-5-isothiocyanate, covalently bound at the enzyme's putative ATP binding site domain, indicated that a distance of about 85 A separates these two regions and that this distance is reduced upon divalent cation binding and increased upon the Na+E1-->K+E2 conformational transition. These data suggest a model for the localization of the terminal moieties of the oligosaccharides that places them, on average, about 18 A from the AO binding site and this distance or less from the extracellular membrane surface.

Full text

PDF
182

Images in this article

191FIGURE 5
on p.191

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amler E., Abbott A., Ball W. J., Jr Structural dynamics and oligomeric interactions of Na+,K(+)-ATPase as monitored using fluorescence energy transfer. Biophys J. 1992 Feb;61(2):553–568. doi: 10.1016/S0006-3495(92)81859-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arystarkhova E., Gibbons D. L., Sweadner K. J. Topology of the Na,K-ATPase. Evidence for externalization of a labile transmembrane structure during heating. J Biol Chem. 1995 Apr 14;270(15):8785–8796. doi: 10.1074/jbc.270.15.8785. [DOI] [PubMed] [Google Scholar]
  3. Brown T. A., Horowitz B., Miller R. P., McDonough A. A., Farley R. A. Molecular cloning and sequence analysis of the (Na+ + K+)-ATPase beta subunit from dog kidney. Biochim Biophys Acta. 1987 Apr 8;912(2):244–253. doi: 10.1016/0167-4838(87)90095-1. [DOI] [PubMed] [Google Scholar]
  4. Carilli C. T., Farley R. A., Perlman D. M., Cantley L. C. The active site structure of Na+- and K+-stimulated ATPase. Location of a specific fluorescein isothiocyanate reactive site. J Biol Chem. 1982 May 25;257(10):5601–5606. [PubMed] [Google Scholar]
  5. Chow D. C., Forte J. G. Functional significance of the beta-subunit for heterodimeric P-type ATPases. J Exp Biol. 1995 Jan;198(Pt 1):1–17. doi: 10.1242/jeb.198.1.1. [DOI] [PubMed] [Google Scholar]
  6. Collins J. H., Leszyk J. The "gamma subunit" of Na,K-ATPase: a small, amphiphilic protein with a unique amino acid sequence. Biochemistry. 1987 Dec 29;26(26):8665–8668. doi: 10.1021/bi00400a026. [DOI] [PubMed] [Google Scholar]
  7. Cornelius F., Skou J. C. Na+-Na+ exchange mediated by (Na+ + K+)-ATPase reconstituted into liposomes. Evaluation of pump stoichiometry and response to ATP and ADP. Biochim Biophys Acta. 1985 Aug 27;818(2):211–221. doi: 10.1016/0005-2736(85)90572-3. [DOI] [PubMed] [Google Scholar]
  8. Eakle K. A., Kim K. S., Kabalin M. A., Farley R. A. High-affinity ouabain binding by yeast cells expressing Na+, K(+)-ATPase alpha subunits and the gastric H+, K(+)-ATPase beta subunit. Proc Natl Acad Sci U S A. 1992 Apr 1;89(7):2834–2838. doi: 10.1073/pnas.89.7.2834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eakle K. A., Lyu R. M., Farley R. A. The influence of beta subunit structure on the interaction of Na+/K(+)-ATPase complexes with Na+. A chimeric beta subunit reduces the Na+ dependence of phosphoenzyme formation from ATP. J Biol Chem. 1995 Jun 9;270(23):13937–13947. doi: 10.1074/jbc.270.23.13937. [DOI] [PubMed] [Google Scholar]
  10. Esmann M. Oligomycin interaction with Na,K-ATPase: oligomycin binding and dissociation are slow processes. Biochim Biophys Acta. 1991 Apr 26;1064(1):31–36. doi: 10.1016/0005-2736(91)90408-z. [DOI] [PubMed] [Google Scholar]
  11. Fiorini R., Valentino M., Wang S., Glaser M., Gratton E. Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles. Biochemistry. 1987 Jun 30;26(13):3864–3870. doi: 10.1021/bi00387a019. [DOI] [PubMed] [Google Scholar]
  12. Fortes P. A. A fluorometric method for the determination of functional (Na,K)-ATPase and cardiac glycoside receptors. Anal Biochem. 1986 Nov 1;158(2):454–462. doi: 10.1016/0003-2697(86)90575-0. [DOI] [PubMed] [Google Scholar]
  13. Geering K. Posttranslational modifications and intracellular transport of sodium pumps: importance of subunit assembly. Soc Gen Physiol Ser. 1991;46:31–43. [PubMed] [Google Scholar]
  14. Geering K., Theulaz I., Verrey F., Häuptle M. T., Rossier B. C. A role for the beta-subunit in the expression of functional Na+-K+-ATPase in Xenopus oocytes. Am J Physiol. 1989 Nov;257(5 Pt 1):C851–C858. doi: 10.1152/ajpcell.1989.257.5.C851. [DOI] [PubMed] [Google Scholar]
  15. Hebert H., Skriver E., Kavéus U., Maunsbach A. B. Coexistence of different forms of Na,K-ATPase in two-dimensional membrane crystals. FEBS Lett. 1990 Jul 30;268(1):83–87. doi: 10.1016/0014-5793(90)80978-r. [DOI] [PubMed] [Google Scholar]
  16. Herbert H., Skriver E., Maunsbach A. B. Three-dimensional structure of renal Na,K-ATPase determined by electron microscopy of membrane crystals. FEBS Lett. 1985 Jul 22;187(1):182–186. doi: 10.1016/0014-5793(85)81238-2. [DOI] [PubMed] [Google Scholar]
  17. Horowitz B., Eakle K. A., Scheiner-Bobis G., Randolph G. R., Chen C. Y., Hitzeman R. A., Farley R. A. Synthesis and assembly of functional mammalian Na,K-ATPase in yeast. J Biol Chem. 1990 Mar 15;265(8):4189–4192. [PubMed] [Google Scholar]
  18. Jaisser F., Canessa C. M., Horisberger J. D., Rossier B. C. Primary sequence and functional expression of a novel ouabain-resistant Na,K-ATPase. The beta subunit modulates potassium activation of the Na,K-pump. J Biol Chem. 1992 Aug 25;267(24):16895–16903. [PubMed] [Google Scholar]
  19. Jørgensen P. L. Structure, function and regulation of Na,K-ATPase in the kidney. Kidney Int. 1986 Jan;29(1):10–20. doi: 10.1038/ki.1986.3. [DOI] [PubMed] [Google Scholar]
  20. Kawamura M., Nagano K. Evidence for essential disulfide bonds in the beta-subunit of (Na+ + K+)-ATPase. Biochim Biophys Acta. 1984 Jul 25;774(2):188–192. doi: 10.1016/0005-2736(84)90290-6. [DOI] [PubMed] [Google Scholar]
  21. Kirley T. L. Determination of three disulfide bonds and one free sulfhydryl in the beta subunit of (Na,K)-ATPase. J Biol Chem. 1989 May 5;264(13):7185–7192. [PubMed] [Google Scholar]
  22. Kirley T. L. Inactivation of (Na+,K+)-ATPase by beta-mercaptoethanol. Differential sensitivity to reduction of the three beta subunit disulfide bonds. J Biol Chem. 1990 Mar 15;265(8):4227–4232. [PubMed] [Google Scholar]
  23. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  24. Lakowicz J. R., Gryczynski I., Cheung H. C., Wang C. K., Johnson M. L., Joshi N. Distance distributions in proteins recovered by using frequency-domain fluorometry. Applications to troponin I and its complex with troponin C. Biochemistry. 1988 Dec 27;27(26):9149–9160. doi: 10.1021/bi00426a012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lakowicz J. R., Jayaweera R., Joshi N., Gryczynski I. Correction for contaminant fluorescence in frequency-domain fluorometry. Anal Biochem. 1987 Feb 1;160(2):471–479. doi: 10.1016/0003-2697(87)90078-9. [DOI] [PubMed] [Google Scholar]
  26. Lakowicz J. R., Laczko G., Cherek H., Gratton E., Limkeman M. Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data. Biophys J. 1984 Oct;46(4):463–477. doi: 10.1016/S0006-3495(84)84043-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lane L. K., Potter J. D., Collins J. H. Large-scale purification of Na,K-ATPase and its protein subunits from lamb kidney medulla. Prep Biochem. 1979;9(2):157–170. doi: 10.1080/00327487908061681. [DOI] [PubMed] [Google Scholar]
  28. Lee J. A., Fortes P. A. Labeling of the glycoprotein subunit of (Na,K)ATPase with fluorescent probes. Biochemistry. 1985 Jan 15;24(2):322–330. doi: 10.1021/bi00323a013. [DOI] [PubMed] [Google Scholar]
  29. Lee J. A., Fortes P. A. Spatial relationship and conformational changes between the cardiac glycoside site and beta-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase. Biochemistry. 1986 Dec 16;25(25):8133–8141. doi: 10.1021/bi00373a002. [DOI] [PubMed] [Google Scholar]
  30. Lutsenko S., Kaplan J. H. An essential role for the extracellular domain of the Na,K-ATPase beta-subunit in cation occlusion. Biochemistry. 1993 Jul 6;32(26):6737–6743. doi: 10.1021/bi00077a029. [DOI] [PubMed] [Google Scholar]
  31. Noguchi S., Mishina M., Kawamura M., Numa S. Expression of functional (Na+ + K+)-ATPase from cloned cDNAs. FEBS Lett. 1987 Dec 10;225(1-2):27–32. doi: 10.1016/0014-5793(87)81125-0. [DOI] [PubMed] [Google Scholar]
  32. Ohta T., Yoshida M., Nagano K., Hirano H., Kawamura M. Structure of the extra-membranous domain of the beta-subunit of (Na,K)-ATPase revealed by the sequences of its tryptic peptides. FEBS Lett. 1986 Aug 18;204(2):297–301. doi: 10.1016/0014-5793(86)80832-8. [DOI] [PubMed] [Google Scholar]
  33. Plesner L., Plesner I. W. Kinetics of oligomycin inhibition and activation of Na+/K(+)-ATPase. Biochim Biophys Acta. 1991 Feb 15;1076(3):421–426. doi: 10.1016/0167-4838(91)90486-j. [DOI] [PubMed] [Google Scholar]
  34. Price E. M., Lingrel J. B. Structure-function relationships in the Na,K-ATPase alpha subunit: site-directed mutagenesis of glutamine-111 to arginine and asparagine-122 to aspartic acid generates a ouabain-resistant enzyme. Biochemistry. 1988 Nov 1;27(22):8400–8408. doi: 10.1021/bi00422a016. [DOI] [PubMed] [Google Scholar]
  35. Schwartz A., Allen J. C., Harigaya S. Possible involvement of cardiac Na+, K+-adenosine triphosphatase in the mechanism of action of cardiac glycosides. J Pharmacol Exp Ther. 1969 Jul;168(1):31–41. [PubMed] [Google Scholar]
  36. Shull G. E., Lane L. K., Lingrel J. B. Amino-acid sequence of the beta-subunit of the (Na+ + K+)ATPase deduced from a cDNA. Nature. 1986 May 22;321(6068):429–431. doi: 10.1038/321429a0. [DOI] [PubMed] [Google Scholar]
  37. Stewart W. W. Functional connections between cells as revealed by dye-coupling with a highly fluorescent naphthalimide tracer. Cell. 1978 Jul;14(3):741–759. doi: 10.1016/0092-8674(78)90256-8. [DOI] [PubMed] [Google Scholar]
  38. Sun Y., Ball W. J., Jr Identification of antigenic sites on the Na+/K(+)-ATPase beta-subunit: their sequences and the effects of thiol reduction upon their structure. Biochim Biophys Acta. 1994 Aug 17;1207(2):236–248. doi: 10.1016/0167-4838(94)00074-3. [DOI] [PubMed] [Google Scholar]
  39. Tamkun M. M., Fambrough D. M. The (Na+ + K+)-ATPase of chick sensory neurons. Studies on biosynthesis and intracellular transport. J Biol Chem. 1986 Jan 25;261(3):1009–1019. [PubMed] [Google Scholar]
  40. Taniguchi K., Mårdh S. Reversible changes in the fluorescence energy transfer accompanying formation of reaction intermediates in probe-labeled (Na+,K+)-ATPase. J Biol Chem. 1993 Jul 25;268(21):15588–15594. [PubMed] [Google Scholar]
  41. Taniguchi K., Sasaki T., Shinoguchi E., Kamo Y., Ito E. Conformational change accompanying formation of oligomycin-induced Na(+)-bound forms and their conversion to ADP-sensitive phosphoenzymes in Na+,K(+)-ATPase. J Biochem. 1991 Feb;109(2):299–306. [PubMed] [Google Scholar]
  42. Treuheit M. J., Costello C. E., Kirley T. L. Structures of the complex glycans found on the beta-subunit of (Na,K)-ATPase. J Biol Chem. 1993 Jul 5;268(19):13914–13919. [PubMed] [Google Scholar]
  43. Zamofing D., Rossier B. C., Geering K. Role of the Na,K-ATPase beta-subunit in the cellular accumulation and maturation of the enzyme as assessed by glycosylation inhibitors. J Membr Biol. 1988 Aug;104(1):69–79. doi: 10.1007/BF01871903. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES