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Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca2+-ATPase by Competitive Inhibition of [γ-32P]TNP-8N3-ATP Photolabeling

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P-Type ATPases

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1377))

Abstract

The photoactivation of aryl azides is commonly employed as a means to covalently attach cross-linking and labeling reagents to proteins, facilitated by the high reactivity of the resultant aryl nitrenes with amino groups present in the protein side chains. We have developed a simple and reliable assay for the determination of the ATP binding affinity of native or recombinant sarcoplasmic reticulum Ca2+-ATPase, taking advantage of the specific photolabeling of Lys492 in the Ca2+-ATPase by [γ-32P]2′,3′-O-(2,4,6-trinitrophenyl)-8-azido-adenosine 5′-triphosphate ([γ-32P]TNP-8N3-ATP) and the competitive inhibition by ATP of the photolabeling reaction. The method allows determination of the ATP affinity of Ca2+-ATPase mutants expressed in mammalian cell culture in amounts too minute for conventional equilibrium binding studies. Here, we describe the synthesis and purification of the [γ-32P]TNP-8N3-ATP photolabel, as well as its application in ATP affinity measurements.

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References

  1. Lacapere JJ, Bennett N, Dupont Y, Guillain F (1990) pH and magnesium dependence of ATP binding to sarcoplasmic reticulum ATPase. Evidence that the catalytic ATP-binding site consists of two domains. J Biol Chem 265(1):348–353

    PubMed  CAS  Google Scholar 

  2. Lacapere JJ, Guillain F (1993) The reaction mechanism of Ca(2+)-ATPase of sarcoplasmic reticulum. Direct measurement of the Mg.ATP dissociation constant gives similar values in the presence or absence of calcium. Eur J Biochem 211(1-2):117–126

    Article  PubMed  CAS  Google Scholar 

  3. Norby JG, Jensen J (1971) Binding of ATP to brain microsomal ATPase. Determination of the ATP-binding capacity and the dissociation constant of the enzyme-ATP complex as a function of K+ concentration. Biochim Biophys Acta 233(1):104–116

    Article  PubMed  CAS  Google Scholar 

  4. Hegyvary C, Post RL (1971) Binding of adenosine triphosphate to sodium and potassium ion-stimulated adenosine triphosphatase. J Biol Chem 246(17):5234–5240

    PubMed  CAS  Google Scholar 

  5. Fedosova NU, Champeil P, Esmann M (2003) Rapid filtration analysis of nucleotide binding to Na,K-ATPase. Biochemistry 42(12):3536–3543

    Article  PubMed  CAS  Google Scholar 

  6. Vilsen B, Andersen JP, MacLennan DH (1991) Functional consequences of alterations to amino acids located in the hinge domain of the Ca(2+)-ATPase of sarcoplasmic reticulum. J Biol Chem 266(24):16157–16164

    PubMed  CAS  Google Scholar 

  7. Sorensen T, Vilsen B, Andersen JP (1997) Mutation Lys758 → Ile of the sarcoplasmic reticulum Ca2+-ATPase enhances dephosphorylation of E2P and inhibits the E2 to E1Ca2 transition. J Biol Chem 272(48):30244–30253

    Article  PubMed  CAS  Google Scholar 

  8. Sorensen TL, Dupont Y, Vilsen B, Andersen JP (2000) Fast kinetic analysis of conformational changes in mutants of the Ca(2+)-ATPase of sarcoplasmic reticulum. J Biol Chem 275(8):5400–5408

    Article  PubMed  CAS  Google Scholar 

  9. Vilsen B (1993) Glutamate 329 located in the fourth transmembrane segment of the alpha-subunit of the rat kidney Na+,K+-ATPase is not an essential residue for active transport of sodium and potassium ions. Biochemistry 32(48):13340–13349

    Article  PubMed  CAS  Google Scholar 

  10. McIntosh DB, Woolley DG, Vilsen B, Andersen JP (1996) Mutagenesis of segment 487Phe-Ser-Arg-Asp-Arg-Lys492 of sarcoplasmic reticulum Ca2+-ATPase produces pumps defective in ATP binding. J Biol Chem 271(42):25778–25789

    Article  PubMed  CAS  Google Scholar 

  11. Seebregts CJ, McIntosh DB (1989) 2′,3′-O-(2,4,6-Trinitrophenyl)-8-azido-adenosine mono-, di-, and triphosphates as photoaffinity probes of the Ca2+-ATPase of sarcoplasmic reticulum. Regulatory/superfluorescent nucleotides label the catalytic site with high efficiency. J Biol Chem 264(4):2043–2052

    PubMed  CAS  Google Scholar 

  12. Chowdhry V, Westheimer FH (1979) Photoaffinity labeling of biological systems. Annu Rev Biochem 48:293–325

    Article  PubMed  CAS  Google Scholar 

  13. Kotzyba-Hibert F, Kapfer I, Goeldner M (1995) Recent trends in photoaffinity labeling. Angew Chem Int Ed Engl 34:1296–1312

    Article  CAS  Google Scholar 

  14. Potter RL, Haley BE (1983) Photoaffinity labeling of nucleotide binding sites with 8-azidopurine analogs: techniques and applications. Methods Enzymol 91:613–633

    Article  PubMed  CAS  Google Scholar 

  15. Suzuki H, Kubota T, Kubo K, Kanazawa T (1990) Existence of a low-affinity ATP-binding site in the unphosphorylated Ca2(+)-ATPase of sarcoplasmic reticulum vesicles: evidence from binding of 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene)-[3H]AMP and -[3H]ATP. Biochemistry 29(30):7040–7045

    Article  PubMed  CAS  Google Scholar 

  16. Clausen JD, McIntosh DB, Woolley DG, Andersen JP (2011) Modulatory ATP binding affinity in intermediate states of E2P dephosphorylation of sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 286(13):11792–11802

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  17. McIntosh DB, Woolley DG, Berman MC (1992) 2′,3′-O-(2,4,6-Trinitrophenyl)-8-azido-AMP and -ATP photolabel Lys-492 at the active site of sarcoplasmic reticulum Ca(2+)-ATPase. J Biol Chem 267(8):5301–5309

    PubMed  CAS  Google Scholar 

  18. McIntosh DB, Woolley DG (1994) Catalysis of an ATP analogue untethered and tethered to lysine 492 of sarcoplasmic reticulum Ca(2+)-ATPase. J Biol Chem 269(34):21587–21595

    PubMed  CAS  Google Scholar 

  19. McIntosh DB, Woolley DG, MacLennan DH, Vilsen B, Andersen JP (1999) Interaction of nucleotides with Asp(351) and the conserved phosphorylation loop of sarcoplasmic reticulum Ca(2+)-ATPase. J Biol Chem 274(36):25227–25236

    Article  PubMed  CAS  Google Scholar 

  20. Clausen JD, McIntosh DB, Woolley DG, Andersen JP (2001) Importance of Thr-353 of the conserved phosphorylation loop of the sarcoplasmic reticulum Ca2+-ATPase in MgATP binding and catalytic activity. J Biol Chem 276(38):35741–35750

    Article  PubMed  CAS  Google Scholar 

  21. Clausen JD, McIntosh DB, Vilsen B, Woolley DG, Andersen JP (2003) Importance of conserved N-domain residues Thr441, Glu442, Lys515, Arg560, and Leu562 of sarcoplasmic reticulum Ca2+-ATPase for MgATP binding and subsequent catalytic steps. Plasticity of the nucleotide-binding site. J Biol Chem 278(22):20245–20258

    Article  PubMed  CAS  Google Scholar 

  22. McIntosh DB, Clausen JD, Woolley DG, MacLennan DH, Vilsen B, Andersen JP (2003) ATP binding residues of sarcoplasmic reticulum Ca(2+)-ATPase. Ann N Y Acad Sci 986:101–105

    Article  PubMed  CAS  Google Scholar 

  23. McIntosh DB, Clausen JD, Woolley DG, MacLennan DH, Vilsen B, Andersen JP (2004) Roles of conserved P domain residues and Mg2+ in ATP binding in the ground and Ca2+-activated states of sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 279(31):32515–32523

    Article  PubMed  CAS  Google Scholar 

  24. Clausen JD, McIntosh DB, Woolley DG, Anthonisen AN, Vilsen B, Andersen JP (2006) Asparagine 706 and glutamate 183 at the catalytic site of sarcoplasmic reticulum Ca2+-ATPase play critical but distinct roles in E2 states. J Biol Chem 281(14):9471–9481

    Article  PubMed  CAS  Google Scholar 

  25. Clausen JD, McIntosh DB, Anthonisen AN, Woolley DG, Vilsen B, Andersen JP (2007) ATP-binding modes and functionally important interdomain bonds of sarcoplasmic reticulum Ca2+-ATPase revealed by mutation of glycine 438, glutamate 439, and arginine 678. J Biol Chem 282(28):20686–20697

    Article  PubMed  CAS  Google Scholar 

  26. Clausen JD, McIntosh DB, Woolley DG, Andersen JP (2008) Critical interaction of actuator domain residues arginine 174, isoleucine 188, and lysine 205 with modulatory nucleotide in sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 283(51):35703–35714

    Article  PubMed  CAS  Google Scholar 

  27. Clausen JD, Bublitz M, Arnou B, Montigny C, Jaxel C, Moller JV, Nissen P, Andersen JP, le Maire M (2013) SERCA mutant E309Q binds two Ca(2+) ions but adopts a catalytically incompetent conformation. EMBO J 32(24):3231–3243

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  28. Clausen JD, Anthonisen AN, Andersen JP (2014) Critical role of interdomain interactions for modulatory ATP binding to sarcoplasmic reticulum Ca2+-ATPase. J Biol Chem 289(42):29123–29134

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  29. Sorensen TL, Moller JV, Nissen P (2004) Phosphoryl transfer and calcium ion occlusion in the calcium pump. Science 304(5677):1672–1675

    Article  PubMed  CAS  Google Scholar 

  30. Toyoshima C, Mizutani T (2004) Crystal structure of the calcium pump with a bound ATP analogue. Nature 430(6999):529–535

    Article  PubMed  CAS  Google Scholar 

  31. Jensen AM, Sorensen TL, Olesen C, Moller JV, Nissen P (2006) Modulatory and catalytic modes of ATP binding by the calcium pump. EMBO J 25(11):2305–2314

    Article  PubMed  PubMed Central  Google Scholar 

  32. Olesen C, Picard M, Winther AM, Gyrup C, Morth JP, Oxvig C, Moller JV, Nissen P (2007) The structural basis of calcium transport by the calcium pump. Nature 450(7172):1036–1042

    Article  PubMed  CAS  Google Scholar 

  33. Moczydlowski EG, Fortes PA (1981) Characterization of 2′,3′-O-(2,4,6-trinitrocyclohexadienylidine)adenosine 5′-triphosphate as a fluorescent probe of the ATP site of sodium and potassium transport adenosine triphosphatase. Determination of nucleotide binding stoichiometry and ion-induced changes in affinity for ATP. J Biol Chem 256(5):2346–2356

    PubMed  CAS  Google Scholar 

  34. Moczydlowski EG, Fortes PA (1981) Inhibition of sodium and potassium adenosine triphosphatase by 2′,3′-O-(2,4,6-trinitrocyclohexadienylidene) adenine nucleotides. Implications for the structure and mechanism of the Na:K pump. J Biol Chem 256(5):2357–2366

    PubMed  CAS  Google Scholar 

  35. Faller LD (1989) Competitive binding of ATP and the fluorescent substrate analogue 2′,3′-O-(2,4,6-trinitrophenylcyclohexadienylidine) adenosine 5′-triphosphate to the gastric H+, K+-ATPase: evidence for two classes of nucleotide sites. Biochemistry 28(16):6771–6778

    Article  PubMed  CAS  Google Scholar 

  36. Faller LD (1990) Binding of the fluorescent substrate analogue 2′,3′-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine 5′-triphosphate to the gastric H+,K+-ATPase: evidence for cofactor-induced conformational changes in the enzyme. Biochemistry 29(13):3179–3186

    Article  PubMed  CAS  Google Scholar 

  37. Axelsen KB (2014) The P-type ATPase Database. http://traplabs.dk/patbase/. Accessed 24 Aug 2014

  38. Axelsen KB, Palmgren MG (1998) Evolution of substrate specificities in the P-type ATPase superfamily. J Mol Evol 46(1):84–101

    Article  PubMed  CAS  Google Scholar 

  39. Decottignies A, Grant AM, Nichols JW, de Wet H, McIntosh DB, Goffeau A (1998) ATPase and multidrug transport activities of the overexpressed yeast ABC protein Yor1p. J Biol Chem 273(20):12612–12622

    Article  PubMed  CAS  Google Scholar 

  40. de Wet H, McIntosh DB, Conseil G, Baubichon-Cortay H, Krell T, Jault JM, Daskiewicz JB, Barron D, Di Pietro A (2001) Sequence requirements of the ATP-binding site within the C-terminal nucleotide-binding domain of mouse P-glycoprotein: structure-activity relationships for flavonoid binding. Biochemistry 40(34):10382–10391

    Article  PubMed  Google Scholar 

  41. Maruyama K, MacLennan DH (1988) Mutation of aspartic acid-351, lysine-352, and lysine-515 alters the Ca2+ transport activity of the Ca2+-ATPase expressed in COS-1 cells. Proc Natl Acad Sci U S A 85(10):3314–3318

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  42. Ko YH, Bianchet M, Amzel LM, Pedersen PL (1997) Novel insights into the chemical mechanism of ATP synthase. Evidence that in the transition state the gamma-phosphate of ATP is near the conserved alanine within the P-loop of the beta-subunit. J Biol Chem 272(30):18875–18881

    Article  PubMed  CAS  Google Scholar 

  43. Pick U (1982) The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum. J Biol Chem 257(11):6111–6119

    PubMed  CAS  Google Scholar 

  44. Dupont Y, Bennett N (1982) Vanadate inhibition of the Ca2+-dependent conformational change of the sarcoplasmic reticulum Ca2+-ATPase. FEBS Lett 139(2):237–240

    Article  PubMed  CAS  Google Scholar 

  45. Owens JR, Haley BE (1984) Synthesis and utilization of 8-azidoguanosine 3′-phosphate 5′-[5′-32P]phosphate. Photoaffinity studies on cytosolic proteins of Escherichia coli. J Biol Chem 259(23):14843–14848

    PubMed  CAS  Google Scholar 

  46. Gourdon P, Liu XY, Skjorringe T, Morth JP, Moller LB, Pedersen BP, Nissen P (2011) Crystal structure of a copper-transporting PIB-type ATPase. Nature 475(7354):59–64

    Article  PubMed  CAS  Google Scholar 

  47. Toyoshima C, Yonekura S, Tsueda J, Iwasawa S (2011) Trinitrophenyl derivatives bind differently from parent adenine nucleotides to Ca2+-ATPase in the absence of Ca2+. Proc Natl Acad Sci U S A 108(5):1833–1838

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  48. Sarma RH, Lee CH, Evans FE, Yathindra N, Sundaralingam M (1974) Probing the interrelation between the glycosyl torsion, sugar pucker, and the backbone conformation in C(8) substituted adenine nucleotides by 1H and 1H-(31P) fast Fourier transform nuclear magnetic resonance methods and conformational energy calculations. J Am Chem Soc 96(23):7337–7348

    Article  PubMed  CAS  Google Scholar 

  49. Glynn IM, Chappell JB (1964) A simple method for the preparation of 32-P-labelled adenosine triphosphate of high specific activity. Biochem J 90(1):147–149

    Article  PubMed  CAS  PubMed Central  Google Scholar 

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Acknowledgements

This work was supported by the Lundbeck Foundation and the Centre for Membrane Pumps in Cells and Disease—PUMPKIN, Danish National Research Foundation (to JDC), the National Research Foundation, South Africa, and the University of Cape Town, South Africa (to DBM and DGW), and the Danish Medical Research Council and the Novo Nordisk Foundation (to JPA).

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Authors and Affiliations

  1. Department of Biomedicine, Aarhus University, Ole Worms Allé 4, Building 1160, 8000, Aarhus C, Denmark

    Johannes D. Clausen & Jens Peter Andersen

  2. Institute of Infectious Diseases and Molecular Medicine, Division of Chemical Pathology, Faculty of Health Sciences, University of Cape Town, Observatory, Cape Town, South Africa

    David B. McIntosh & David G. Woolley

Authors
  1. Johannes D. Clausen
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  2. David B. McIntosh
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  3. David G. Woolley
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  4. Jens Peter Andersen
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Corresponding author

Correspondence to Johannes D. Clausen .

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Editors and Affiliations

  1. Department of Biochemistry, Biocenter, University of Oxford, Oxford, Oxfordshire, United Kingdom

    Maike Bublitz

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Clausen, J.D., McIntosh, D.B., Woolley, D.G., Andersen, J.P. (2016). Determination of the ATP Affinity of the Sarcoplasmic Reticulum Ca2+-ATPase by Competitive Inhibition of [γ-32P]TNP-8N3-ATP Photolabeling. 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_22

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  • DOI: https://doi.org/10.1007/978-1-4939-3179-8_22

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3178-1

  • Online ISBN: 978-1-4939-3179-8

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