Abstract
Plasma membrane Ca2+ ATPases (PMCA pumps) are key regulators of cytosolic Ca2+ in eukaryotes. They extrude Ca2+ from the cytosol, using the energy of ATP hydrolysis and operate as Ca2+-H+ exchangers. They are activated by the Ca2+-binding protein calmodulin, by acidic phospholipids and by other mechanisms, among them kinase-mediated phosphorylation. Isolation of the PMCA in pure and active form is essential for the analysis of its structure and function. In this chapter, the purification of the pump, as first achieved from erythrocyte plasma membranes by calmodulin-affinity chromatography, is described in detail. The reversible, high-affinity, Ca2+-dependent interaction of the pump with calmodulin is the basis of the procedure. Either phospholipids or glycerol have to be present in the isolation buffers to keep the pump active during the isolation procedure. After the isolation of the PMCA pump from human erythrocytes the pump was purified from other cell types, e.g., heart sarcolemma, plant microsomal fractions, and cells that express it ectopically. The reconstitution of the purified pump into phospholipid vesicles using the cholate dialysis method will also be described. It allows studies of transport mechanism and of regulation of pump activity. The purified pump can be stored in the reconstituted form for several days at 4 °C with little loss of activity, but it rapidly loses activity when stored in the detergent-solubilized form.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Brini M, Cali T, Ottolini D, Carafoli E (2013) The plasma membrane Ca2+ pump in health and disease. FEBS J 280:5385–5397
Bonza MC, Morandini P, Luoni L (2000) At-ACA8 encodes a plasma membrane-localized Ca2+-ATPase of Arabidopsis with a calmodulin-binding domain at the N Terminus. Plant Physiol 123:1495–1505
Niggli V, Ronner P, Carafoli C, Penniston JT (1979) Effects of calmodulin on the (Ca2+-Mg2+)-ATPase partially purified from erythrocyte membranes. Arch Biochem Biophys 198:124–130
Niggli V, Penniston JT, Carafoli E (1979) Purification of the (Ca2+-Mg2+)-ATPase from human erythrocyte membranes using a calmodulin affinity column. J Biol Chem 254:9955–9958
Niggli V, Sigel E, Carafoli E (1982) The purified Ca2+ pump of human erythrocyte membranes catalyzes an electroneutral Ca2+-H+ exchange in reconstituted liposomal systems. J Biol Chem 257:2350–2356
Waldeck AR, Xu AS, Roufogalis BD, Kuchel PV (1998) Measurements of Ca2+ and H+ transport mediated by A23187 and reconstituted plasma membrane Ca2+-ATPase. Eur Biophys J 27:255–262
Thomas RC (2009) The plasma membrane calcium ATPase (PMCA) of neurons is electroneutral and exchanges 2 H+ for each Ca2+ or Ba2+ ion extruded. J Physiol 587:315–327
Hao L, Rigaud JL, Inesi G (1994) Ca2+/H+ countertransport and electrogenicity in proteoliposomes containing erythrocyte plasma membrane Ca-ATPase and exogenous lipids. J Biol Chem 269:14265–14275
Niggli V, Sigel E (2008) Anticipating antiport in P-type ATPases. Trends Biochem Sci 33:156–160
Niggli V, Adunyah ES, Penniston JT, Carafoli E (1981) Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane: reconstitution and effect of calmodulin and phospholipids. J Biol Chem 256:395–401
Niggli V, Zurini M, Carafoli E (1987) Purification, reconstitution and molecular characterization of the Ca2+ pump of plasma membranes. Methods Enzymol 139:791–808
Graf E, Verma AK, Gorski JP, Lopaschuk G, Niggli V, Zurini M et al (1982) Molecular properties of Ca2+ pumping ATPase from human erythrocytes. Biochemistry 21:4511–4516
Stauffer TP, Guerini D, Carafoli E (1995) Tissue distribution of the four gene products of the plasma membrane Ca2+ pump. J Biol Chem 270:12184–12190
Niggli V, Adunyah ES, Cameron BF, Bababunmi EA, Carafoli E (1982) The Ca2+-pump of sickle cell plasma membranes. Purification and reconstitution of the ATPase enzyme. Cell Calcium 3:131–151
Caroni P, Zurini M, Clark A, Carafoli E (1983) Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma. J Biol Chem 258:7305–7310
Filomatori CV, Rega AF (2003) On the mechanism of activation of the plasma membrane Ca2+-ATPase by ATP and acidic phospholipids. J Biol Chem 278:22265–22271
Lopreiato R, Giacomello M, Carafoli E (2014) The plasma membrane calcium pump: new ways to look at an old enzyme. J Biol Chem 289:10261–10268
Bonza C, Carnelli A, De Michelis MI, Rasi-Caldogno F (1998) Purification of the plasma membrane Ca2+-ATPase from radish seedlings by calmodulin-agarose affinity chromatography. Plant Physiol 116:845–851
Heim R, Iwata T, Zvaritch E, Adamo HP, Rutishauser B, Strehler EE, Guerini D, Carafoli E (1992) Expression, purification and properties of the plasma membrane Ca2+ pump and of its N-terminally truncated 105-kDa fragment. J Biol Chem 267:24476–24484
Cura CI, Corradi GR, Rinaldi DE, Adamo HP (2008) High sensibility to reactivation by acidic lipids of the recombinant human plasma membrane Ca2+-ATPase isoform 4xb purified from Saccharomyces cerevisiae. Biochim Biophys Acta 1778:2757–2764
Lotersztain S, Hanoune J, Pecker F (1981) A high affinity calcium-stimulated magnesium-dependent ATPase in rat liver plasma membranes. J Biol Chem 256:11209–11215
Penniston JT, Filoteo AG, McDonough CS, Carafoli E (1988) Purification, reconstitution and regulation of plasma membrane Ca2+-pumps. Methods Enzymol 157:340–351
Saimi S, Kung C (2002) Calmodulin as an ion channel subunit. Annu Rev Physiol 64:289–311
Klaerke DA (1995) Purification and characterization of epithelial Ca2+-activated K+ channel proteins by calmodulin. Kidney Int 48:1047–1056
Vorherr T, Kessler T, Hofmann F, Carafoli E (1991) The calmodulin-binding domain mediates the self-association of the plasma membrane Ca2+ pump. J Biol Chem 266:22–27
Kosk-Kosicka D, Bzdega T (1988) Activation of the erythrocyte Ca2+ ATPase by either self-association or interaction with calmodulin. J Biol Chem 263:18184–18189
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Niggli, V., Carafoli, E. (2016). The Plasma Membrane Ca2+ ATPase: Purification by Calmodulin Affinity Chromatography, and Reconstitution of the Purified Protein. In: Bublitz, M. (eds) P-Type ATPases. Methods in Molecular Biology, vol 1377. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3179-8_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3179-8_7
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3178-1
Online ISBN: 978-1-4939-3179-8
eBook Packages: Springer Protocols