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Solubilization, purification, and reconstitution of α2β1 isozyme of Na+/K+-ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C12E8, and Triton X-100

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Abstract

We identified α2, α1, and β1 isoforms of Na+/K+-ATPase in caveolae vesicles of bovine pulmonary smooth muscle plasma membrane. The biochemical and biophysical characteristics of the α2β1 isozyme of Na+/K+-ATPase from caveolae vesicles were studied during solubilization and purification using the detergents 1,2-heptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C12E8), and Triton X-100, and reconstitution with the phospholipid dioleoyl-phosphatidylcholine (DOPC). DHPC was determined to be superior to C12E8, whereas C12E8 was better than Triton X-100 in the active enzyme yields and specific activity. Fluorescence studies with DHPC-purified α2β1 isozyme of Na+/K+-ATPase elicited higher E1Na−E2 K transition compared with that of the C12E8- and Triton X-100-purified enzyme. The rate of Na+ efflux in DHPC–DOPC-reconstituted isozyme was higher compared to the C12E8–DOPC- and Triton X100–DOPC-reconstituted enzyme. Circular dichroism analysis suggests that the DHPC-purified α2β1 isozyme of Na+/K+-ATPase possessed more organized secondary structure compared to the C12E8- and Triton X-100-purified isozyme.

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References

  1. Jorgensen PL (1982) Mechanism of the Na+, K+ pump. Protein structure and conformations of the pure (Na+-K+)-ATPase. Biochim Biophys Acta 694:27–68

    PubMed  CAS  Google Scholar 

  2. Skou JC, Esmann M (1992) The Na, K-ATPase. J Bioenerg Biomembr 24:249–261

    PubMed  CAS  Google Scholar 

  3. Pressley TA (1996) Structure and function of the Na, K pump: ten years of molecular biology. Miner Electrolyte Metab 22:264–271

    PubMed  CAS  Google Scholar 

  4. Jorgensen PL, Nielsen JM, Rasmussen JH, Pedersen PA (1998) Structure-function relationships based on ATP binding and cation occlusion at equilibrium in Na+/K+-ATPase. Acta Physiol Scand Suppl 643:79–87

    PubMed  CAS  Google Scholar 

  5. Orlowski J, Lingrel JB (1988) Tissue-specific and developmental regulation of rat Na, K-ATPase catalytic alpha isoform and beta subunit mRNAs. J Biol Chem 263:10436–10442

    PubMed  CAS  Google Scholar 

  6. Juhaszova M, Blaustein MP (1997) Na+ pump low and high ouabain affinity alpha subunit isoforms are differently distributed in cells. Proc Natl Acad Sci USA 94:1800–1805. doi:10.1073/pnas.94.5.1800

    Article  PubMed  CAS  Google Scholar 

  7. Juhaszova M, Blaustein MP (1997) Distinct distribution of different Na+ pump alpha subunit isoforms in plasmalemma. Physiological implications. Ann NY Acad Sci 834:524–536. doi:10.1111/j.1749-6632.1997.tb52310.x

    Article  PubMed  CAS  Google Scholar 

  8. Daniel EE, El-Yazbi A, Cho WJ (2006) Caveolae and calcium handling, a review and a hypothesis. J Cell Mol Med 10:529–544. doi:10.1111/j.1582-4934.2006.tb00418.x

    Article  PubMed  CAS  Google Scholar 

  9. Shaul PW, Anderson RG (1998) Role of plasmalemmal caveolae in signal transduction. Am J Physiol 275:L843–L851

    PubMed  CAS  Google Scholar 

  10. Dostanic I, Paul RJ, Lorenz JN, Theriault S, Van Huysse JW, Lingrel JB (2005) The alpha2-isoform of Na-K-ATPase mediates ouabain-induced hypertension in mice and increased vascular contractility in vitro. Am J Physiol Heart Circ Physiol 288:H477–H485. doi:10.1152/ajpheart.00083.2004

    Article  PubMed  CAS  Google Scholar 

  11. James PF, Grupp IL, Grupp G, Woo AL, Askew GR, Croyle ML, Walsh RA, Lingrel JB (1999) Identification of a specific role for the Na, K, -ATPase alpha-2 isoform as regulator of calcium in the heart. Mol Cell 3:555–563. doi:10.1016/S1097-2765(00)80349-4

    Article  PubMed  CAS  Google Scholar 

  12. Dostanic I, Lorenz JN, Schultz Jel J, Grupp IL, Neumann JC, Wani MA, Lingrel JB (2003) The alpha2 isoform of Na, K-ATPase mediates ouabain-induced cardiac inotropy in mice. J Biol Chem 278:53026–53034. doi:10.1074/jbc.M308547200

    Article  PubMed  CAS  Google Scholar 

  13. Adir Y, Welch LC, Dumasius V, Factor P, Sznajder JI, Ridge KM (2008) Overexpression of the Na-K-ATPase alpha2-subunit improves lung liquid clearance during ventilation-induced lung injury. Am J Physiol Lung Cell Mol Physiol 294:L1233–L1237. doi:10.1152/ajplung.00076.2007

    Article  PubMed  CAS  Google Scholar 

  14. Jorgensen PL (1974) Purification and characterization of (Na+ plus K+)-ATPase. 3. Purification from the outer medulla of mammalian kidney after selective removal of membrane components by sodium dodecylsulphate. Biochim Biophys Acta 356:36–52. doi:10.1016/0005-2736(74)90292-2

    Article  PubMed  CAS  Google Scholar 

  15. Skou JC, Esmann M (1979) Preparation of membrane-bound and of solubilized (Na+ + K+)-ATPase from rectal glands of Squalus acanthias. The effect of preparative procedures on purity, specific and molar activity. Biochim Biophys Acta 567:436–444

    PubMed  CAS  Google Scholar 

  16. Jorgensen PL, Skou JC (1971) Purification and characterization of (Na+ + K+)-ATPase. I. The influence of detergents on the activity of (Na+ + K+)-ATPase in preparations from the outer medulla of rabbit kidney. Biochim Biophys Acta 233:366–380. doi:10.1016/0005-2736(71)90334-8

    Article  PubMed  CAS  Google Scholar 

  17. Brotherus JR, Jost PC, Griffith OH, Hokin LE (1979) Detergent inactivation of sodium- and potassium-activated adenosinetriphosphatase of the electric eel. Biochemistry 18:5043–5050. doi:10.1021/bi00590a003

    Article  PubMed  CAS  Google Scholar 

  18. Santos HL, Lamas RP, Ciancaglini P (2002) Solubilization of Na, K-ATPase from rabbit kidney outer medulla using only C12E8. Braz J Med Biol Res 35:277–288

    PubMed  CAS  Google Scholar 

  19. Jackson RL, Verkleij AJ, van Zoelen EJ, Lane LK, Schwartz A, van Deenen LL (1980) Asymmetric incorporation of Na+, K+-ATPase into phospholipid vesicles. Arch Biochem Biophys 200:269–278. doi:10.1016/0003-9861(80)90354-9

    Article  PubMed  CAS  Google Scholar 

  20. Morohashi M, Konishi K, Kawamura M (1988) Effects of Na+ and K+ on Artemia salina (Na, K)-ATPase solubilized with a zwitterionic detergent. J Biochem 103:1073–1077

    PubMed  CAS  Google Scholar 

  21. Mandal A, Das S, Chakraborti T, Kar P, Ghosh B, Chakraboti S (2006) Solubilization, purification and reconstitution of Ca2+-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC. Biochim Biophys Acta 1760:20–31

    PubMed  CAS  Google Scholar 

  22. Kessi J, Poiree JC, Wehrli E, Bachofen R, Semenza G, Hauser H (1994) Short-chain phosphatidylcholines as superior detergents in solubilizing membrane proteins and preserving biological activity. Biochemistry 33:10825–10836. doi:10.1021/bi00201a033

    Article  PubMed  CAS  Google Scholar 

  23. Shivanna BD, Rowe ES (1997) Preservation of the native structure and function of Ca2+-ATPase from sarcoplasmic reticulum: solubilization and reconstitution by new short-chain phospholipid detergent 1, 2-diheptanoyl-sn- phosphatidylcholine. Biochem J 325:533–542

    PubMed  CAS  Google Scholar 

  24. Chakraborti T, Ghosh SK, Michael JR, Chakraborti S (1996) Role of approtinin sensitive protease in the activation of Ca2+-ATPase by superoxide radical in microsomes of pulmonary vascular smooth muscle. Biochem J 317:885–890

    PubMed  CAS  Google Scholar 

  25. Mandal M, Mandal A, Das S, Chakraborti S, Chakraborti T (2003) Identification, purification and partial characterization of tissue inhibitor of matrix metalloprotease-2 in bovine pulmonary artery smooth muscle. Mol Cell Biochem 254:275–287. doi:10.1023/A:1027389602772

    Article  PubMed  CAS  Google Scholar 

  26. Ikezu T, Trapp BD, Song KS, Schlega A, Lisanti MP, Okamoto T (1998) Caveolae, plasma membrane microdomains for α-secretase-mediated processing of the amyloid precussor protein. J Biol Chem 273:10485–10495. doi:10.1074/jbc.273.17.10485

    Article  PubMed  CAS  Google Scholar 

  27. Sargiacomo M, Sudol M, Tang ZL, Lisanti MP (1993) Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol 122:789–807. doi:10.1083/jcb.122.4.789

    Article  PubMed  CAS  Google Scholar 

  28. Lisanti MP, Scherer PE, Vidugiriene J, Tang Z, Vosatka AH, Tu YH, Cook RF, Sargiacomo M (1994) Characterization of caveolin reach membrane domains isolated from an endothelial-rich source: Implications for human disease. J Cell Biol 126:111–126. doi:10.1083/jcb.126.1.111

    Article  PubMed  CAS  Google Scholar 

  29. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354. doi:10.1073/pnas.76.9.4350

    Article  PubMed  CAS  Google Scholar 

  30. Chakraborti T, Das S, Chakraborti S (2005) Proteolytic activation of protein kinase C alpha by peroxynitrite in stimulating cytosolic phospholipase A2 in pulmonary endothelium: involvement of a pertussis toxin sensitive protein. Biochemistry 44:5246–5257. doi:10.1021/bi0477889

    Article  PubMed  CAS  Google Scholar 

  31. Lin H, Ozaki S, Fujishiro N, Takeda K, Imanaga I, Prestwich GD, Inoue M (2005) Subunit composition and role of Na+/K+-ATPases in adrenal chromaffin cells. J Physiol 564:161–172. doi:10.1113/jphysiol.2004.081455

    Article  PubMed  CAS  Google Scholar 

  32. Robinson JD (1967) Kinetic studies on a brain microsomal adenosine triphosphatase. Evidence suggesting conformational changes. Biochemistry 6:3250–3258. doi:10.1021/bi00862a034

    Article  PubMed  CAS  Google Scholar 

  33. Fiske CH, Subbaraw Y (1925) The colorimetric determination of Phosphorus. J Biol Chem 66:375–400

    CAS  Google Scholar 

  34. Hubert JJ, Schenk DB, Skelly H, Leffert HL (1986) Rat hepatic (Na+, K+)-ATPase: alpha-subunit isolation by immunoaffinity chromatography and structural analysis by peptide mapping. Biochemistry 25:4156–4163. doi:10.1021/bi00362a025

    Article  PubMed  CAS  Google Scholar 

  35. Deri Z, Adam-Vizi V (1993) Detection of intracellular free Na+ concentration of synaptosomes by a fluorescent indicator, Na+-binding benzofuran isophthalate: the effect of veratridine, ouabain, and α-latrotoxin. J Neurochem 61:818–825. doi:10.1111/j.1471-4159.1993.tb03592.x

    Article  PubMed  CAS  Google Scholar 

  36. Rigos CF, de Lima Santosh H, Thedei G Jr, Ward RJ (2003) Influence of enzyme conformational changes on catalytic activity investigated by circular dichroism spectroscopy. Biochem Mol Biol Educ 31:329–332. doi:10.1002/bmb.2003.494031050264

    Article  CAS  Google Scholar 

  37. Karlish SJD (1980) Characterization of conformational changes in Na+/K+-ATPase labeled with fluorescein at the active site. J Bioenerg Biomembr 12:111–136. doi:10.1007/BF00744678

    Article  PubMed  CAS  Google Scholar 

  38. Komatsu H, Guy PT, Rowe ES (1993) Effect of unilamellar vesicle size on ethanol-induced interdigitation in dipalmitoylphosphatidylcholine. Chem Phys Lipids 65:11–21. doi:10.1016/0009-3084(93)90077-G

    Article  PubMed  CAS  Google Scholar 

  39. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85. doi:10.1016/0003-2697(85)90442-7

    Article  PubMed  CAS  Google Scholar 

  40. Daniel WW (1978) Biostatistics. In: Daniel WW (ed) A foundation for analysis in the health sciences (Estimation). Wiley, New York, pp 121–157

    Google Scholar 

  41. Juan G, Traganos F, Darzynkiewicz Z (1999) Histone H3 phosphorylation in human monocytes and during HL-60 cell differentiation. Exp Cell Res 246:212–220. doi:10.1006/excr.1998.4283

    Article  PubMed  CAS  Google Scholar 

  42. Garcia M, Bondada V, Geddes JW (2005) Mitochondrial localization of μ calpain. Biochem Biophys Res Commun 338:1241–1247. doi:10.1016/j.bbrc.2005.10.081

    Article  PubMed  CAS  Google Scholar 

  43. Eskelinen EL (2006) Role of LAMP-1 and LAMP-2 in lysosome biogenesis and autophagy. Mol Aspects Med 27:495–502. doi:10.1016/j.mam.2006.08.005

    Article  PubMed  CAS  Google Scholar 

  44. Hood JL, Brooks WH, Roszman TL (2004) Differential compartmentalization of the calpain/calpastatin network with the endoplasmic reticulum and golgi apparatus. J Biol Chem 279:43126–43135. doi:10.1074/jbc.M408100200

    Article  PubMed  CAS  Google Scholar 

  45. Hood JL, Logan BB, Sinai AP, Brooks WH, Roszman TL (2003) Association of the calpain/calpastatin network with subcellular organelles. Biochem Biophys Res Commun 310:1200–1212. doi:10.1016/j.bbrc.2003.09.142

    Article  PubMed  CAS  Google Scholar 

  46. Baer HP, Vriend RA (1985) Cytosolic enzyme leakage from isolated smooth muscle preparations. Can J Physiol Pharmacol 63:164–165

    PubMed  CAS  Google Scholar 

  47. Helenius A, McCaslin DR, Fries E, Tanford C (1979) Properties of detergents. Methods Enzymol 56:734–749. doi:10.1016/0076-6879(79)56066-2

    Article  PubMed  CAS  Google Scholar 

  48. Koepsell H (1986) Methodological aspects of purification and reconstitution of transport proteins from mammalian plasma membranes. Rev Physiol Biochem Pharmacol 104:65–137. doi:10.1007/BFb0031013

    Article  PubMed  CAS  Google Scholar 

  49. Silvius JR (1992) Solubilization and functional reconstitution of biomembrane components. Annu Rev Biophys Biomol Struct 21:323–348. doi:10.1146/annurev.bb.21.060192.001543

    Article  PubMed  CAS  Google Scholar 

  50. Santos HL, Ciancaglini P (2000) A practical approach to the choice of a suitable detergent and optimal conditions for solubilizing a membrane protein. Biochem Educ 28:178–182. doi:10.1016/S0307-4412(99)00107-7

    Article  Google Scholar 

  51. le Maire M, Champeil P, Moller JV (2000) Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 1508:86–111. doi:10.1016/S0304-4157(00)00010-1

    Article  PubMed  Google Scholar 

  52. Tausk RJ, Karmiggelt J, Oudshoorn C, Overbeek JT (1974) Physical chemical studies of short-chain lecithin homologues. I. Influence of the chain length of the fatty acid ester and of electrolytes on the critical micelle concentration. Biophys Chem 1:175–183. doi:10.1016/0301-4622(74)80004-9

    Article  PubMed  CAS  Google Scholar 

  53. Tausk RJ, van Esch J, Karmiggelt J, Voordouw G, Overbeek JT (1974) Physical chemical studies of short-chain lecithin homologues. II. Micellar weights of dihexanoyl- and diheptanoyllecithin. Biophys Chem 1:184–203. doi:10.1016/0301-4622(74)80005-0

    Article  PubMed  CAS  Google Scholar 

  54. Hesketh TR, Smith GA, Houslay MD, McGill KA, Birdsall NJ, Metcalfe JC, Warren GB (1976) Annular lipids determine the ATPase activity of a calcium transport protein complexed with dipalmitoyllecithin. Biochemistry 15:4145–4151. doi:10.1021/bi00664a002

    Article  PubMed  CAS  Google Scholar 

  55. Anholt R, Lindstrom J, Montal M (1981) Stabilization of acetylcholine receptor channels by lipids in cholate solution and during reconstitution in vesicles. J Biol Chem 256:4377–4387

    PubMed  CAS  Google Scholar 

  56. Fukuda K, Ikegami A, Nasuda-Kouyama A, Kouyama T (1990) Effect of partial delipidation of purple membrane on the photodynamics of bacteriorhodopsin. Biochemistry 29:1997–2002. doi:10.1021/bi00460a006

    Article  PubMed  CAS  Google Scholar 

  57. Esmann M (1988) Solubilization of Na+ , K+ -ATPase. Methods Enzymol 156:72–79. doi:10.1016/0076-6879(88)56010-X

    Article  PubMed  CAS  Google Scholar 

  58. Esmann M, Skou JC (1984) Kinetic properties of C12E8-solubilized (Na+-K+)-ATPase. Biochim Biophys Acta 787:71–80

    PubMed  CAS  Google Scholar 

  59. de Foresta B, le Maire M, Orlowski S, Champeil P, Lund S, Moller JV, Michelangeli F, Lee AG (1989) Membrane solubilization by detergent: use of brominated phospholipids to evaluate the detergent-induced changes in Ca2+-ATPase/lipid interaction. Biochemistry 28:2558–2567. doi:10.1021/bi00432a032

    Article  PubMed  Google Scholar 

  60. Ackers GK (1967) Molecular sieve studies of interacting protein systems.I. Equations for transport of associating systems. J Biol Chem 242:3026–3034

    PubMed  CAS  Google Scholar 

  61. Brotherus JR, Jacobsen L, Jorgensen PL (1983) Soluble and enzymatically stable (Na+- K+)-ATPase from mammalian kidney consisting predominantly of protomer alpha beta-units Preparation, assay and reconstitution of active Na+, K+ transport. Biochim Biophys Acta 731:290–303. doi:10.1016/0005-2736(83)90021-4

    Article  PubMed  CAS  Google Scholar 

  62. de Lima Santos H, Lopes ML, Maggio B, Ciancaglini P (2005) Na, K-ATPase reconstituted in liposomes: effects of lipid composition on hydrolytic activity and enzyme orientation. Colloids Surf B Biointerfaces 41:239–248. doi:10.1016/j.colsurfb.2004.12.013

    Article  PubMed  Google Scholar 

  63. Cornelius F (1991) Functional reconstitution of the sodium pump. Kinetics of exchange reactions performed by reconstituted Na/K-ATPase. Biochim Biophys Acta 1071:19–66

    PubMed  CAS  Google Scholar 

  64. Michelangeli F, Munkonge FM (1991) Methods of reconstitution of the purified sarcoplasmic reticulum (Ca2+–Mg2+)-ATPase using bile salt detergents to form membranes of defined lipid to protein ratios or sealed vesicles. Anal Biochem 194:231–236. doi:10.1016/0003-2697(91)90223-G

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

Financial assistance from the Department of Science & Technology (DST), Govt. of India and the Indian Council of Medical Research (ICMR), New Delhi are gratefully acknowledged. Thanks are due to Dr. N. Das and S. N. Dey (Indian Institute of Chemical Biology, Kolkata), Dr. A. N. Ghosh (National Institute of Cholera and Enteric Diseases, Kolkata), and Prof. Kasturi Datta (School of Environmental Sciences, Jawaharlal Nehru University, New Delhi) for their help in our research.

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  1. Department of Biochemistry and Biophysics, University of Kalyani, Kalyani, West Bengal, 741235, India

    Biswarup Ghosh, Tapati Chakraborti, Pulak Kar, Kuntal Dey & Sajal Chakraborti

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  1. Biswarup Ghosh
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Ghosh, B., Chakraborti, T., Kar, P. et al. Solubilization, purification, and reconstitution of α2β1 isozyme of Na+/K+-ATPase from caveolae of pulmonary smooth muscle plasma membrane: comparative studies with DHPC, C12E8, and Triton X-100. Mol Cell Biochem 323, 169–184 (2009). https://doi.org/10.1007/s11010-008-9977-0

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