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Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

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Abstract

The Ca2+/Mg2+ ATPase, which is activated by millimolar concentrations of Ca2+ or Mg2+, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN3, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca2+/Mg2+ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/MgATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca2+/Mg2+ ATPase and was found to bind to the purified enzyme with a KD of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca2+/Mg2+ ATPase is a glycoprotein and contains a large amount of lipids.

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References

  1. Dhalla NS, Zhao D: Cell membrane Ca2+/Mg2+ ATPases. Prog Biophys Mol Biol 52: 1–37, 1989

    Google Scholar 

  2. Dhalla NS, Pierce GN, Panagia V, Singal PK, Beamish RE: Calcium movements in relation to heart function. Basic Res Cardiol 77: 117–139, 1982

    Google Scholar 

  3. Anand MB, Chauhan MS, Dhalla NS: Ca2+/Mg2+ ATPase activities of heart sarcolemma, microsomes and mitochondria. J Biochem (Tokyo) 82: 1731–1739, 1977

    Google Scholar 

  4. McNamara DB, Singh JN, Dhalla NS: Properties of some heart sarcolemmal enzymes. J Biochem (Tokyo) 76: 603–609, 1974

    Google Scholar 

  5. Malouf NN, Meissner G: Cytochemical localization of a ‘basic’ ATPase to canine myocardial surface membrane. J Histochem Cytochem 28: 1286–1294, 1980

    Google Scholar 

  6. Benham CD, Tsien RW: Calcium-permeable channels in vascular smooth muscle. In: LJ Mandel, DC Eaton (eds.) Cell calcium and control of membrane transport. Rockfeller University Press, New York, 1986, pp 46–64

    Google Scholar 

  7. DeYoung MB, Scarpa A: Extracellular ATP induces Ca" transients in cardiac myocytes which are potentiated by norepinephrine. FEBS Lett 223: 53–58, 1987

    Google Scholar 

  8. Hidalgo C, Gonzalez ME, Lagos R: Characterization of the Ca2+ or Mg2+-ATPase of transverse tubule membranes isolated from rabbit skeletal muscle. J Biol Chem 258: 13937–13945, 1983

    Google Scholar 

  9. Kirley TL: Purification and characterization of Mg2+ ATPase from rabbit skeletal muscle transverse tubule. J Biol Chem 263: 12682–12689, 1988

    Google Scholar 

  10. Knowles AF, Leng L: The common occurrence of ATP diphosphohydrolase in mammalian plasma membranes. Biochem Biophys Acta 731: 88–96, 1983

    Google Scholar 

  11. Gantzer ML, Grisham LM: Characterization of Mg2+ AT-Pase from sheep kidney medulla. Arch Biochem Biophys 198:263–269, 1979

    Google Scholar 

  12. Zhao D, Dhalla NS: Influence of gramicidin S on cardiac membrane Ca2+Mg2+ ATPase activities and contractile force development. Can J Physiol Pharmacol 67: 546–552, 1989

    Google Scholar 

  13. Dhalla NS, Ziegelhoffer A, Harrow JAC: Regulatory role of membrane systems in heart function. Can J Physiol Pharmacol 55: 1211–1231, 1977

    Google Scholar 

  14. Harrow JAC, Das PK, Dhalla NS: Influence of some divalent cations on heart sarcolemmal bound enzymes and calcium binding. Biochem Pharmacol 27: 2605–2609, 1978

    Google Scholar 

  15. Ziegelhoffer A, Anand-Srivastava MB, Khandelwal RL, Dhalla NS: Activation of heart sarcolemmal Ca2+/Mg2+ ATPase by cyclic AMP-dependent protein kinase. Biochem Biophys Res Commun 89: 1073–1081, 1979

    Google Scholar 

  16. Ziegelhoffer A, Dhalla NS: Activation of Ca2+/Mg2+ ATPase in heart sarcolemma upon electrical stimulation. Mol Cell Biochem 77: 135–141, 1987

    Google Scholar 

  17. Dhalla NS, Pierce GN, Elimban V: Involvement of the sarcolemmal membrane in cardiac contraction and relaxation. In: H Abe, Y Ito, M Tada, LH Opie (eds.) Regulation of Cardiac Function. Japan Scientific Press, Tokyo, 1984, pp 11–22

    Google Scholar 

  18. Dhalla NS, Smith Cl, Pierce GN, Elimban V, Makino N, Khatter JC: Heart sarcolemmal cation pumps and binding sites. In: H Rupp (ed.) Regulation of Heart Function — Basic Concepts and Clinical Application. Thiame-Stratton, New York, 1985, pp 121–136

    Google Scholar 

  19. Dhalla NS, Anand-Srivastava MB, Tuana BS, Khandelwal RL: Solubilization of calcium dependent adenosine triphosphate from rat heart sarcolemma. J Mol Cell Cardiol 13: 413–423, 1981

    Google Scholar 

  20. Tuana BS, Dhalla NS: Solubilization of a divalent cation dependent ATPase from dog heart sarcolemma. Mol Cell Biochem 77: 79–87, 1987

    Google Scholar 

  21. Tuana BS, Dhalla NS: Purification and characterization of a Ca2+-dependent ATPase from heart sarcolemma. J Biol Chem 257: 14440–14445, 1982

    Google Scholar 

  22. Tuana BS, Dhalla NS: Purification and characterization of a Ca2+/Mg2+ ecto-ATPase from rat heart sarcolemma. Mol Cell Biochem 81: 75–88, 1988

    Google Scholar 

  23. Dhalla NS, Pierce GN: Isolation and characterization of the sarcolemmal membranes from the heart. In: NS Dhalla (ed.) Methods for Studying Cardiac Membranes. CRC Press, Boca Raton, 1984, Vol 1, pp 3–18

    Google Scholar 

  24. Moffat MD, Singal PK, Dhalla NS: Differences in sarcolemmal preparations: Cell surface material and membrane sidedness. Basic Res Cardiol 78: 451–461, 1983

    Google Scholar 

  25. Zhao D, Dhalla NS: Characterization of heart plasma membrane Ca2+/Mg2+ ATPase. Arch Biochem Biophys 263:281–292, 1988

    Google Scholar 

  26. Zhao D, Makino N, Dhalla NS: Specific stimulation of heart sarcolemmal Ca2+/Mg2+ ATPase by concanavalin A. Arch Biochem Biophys 268: 5–40–48, 1989.

    Google Scholar 

  27. Pitts BJR: Stoichiometry of sodium calcium exchange in cardiac sarcolemmal vesicles. J Biol Chem 254: 6232–6234, 1979

    Google Scholar 

  28. Makino N, Dhalla KS, Elimban V, Dhalla NS: Sarcolemmal Ca2+ transport in streptozotocin-induced diabetic cardiomyopathy in rats. Am J Physiol 253: E202-E207, 1987

    Google Scholar 

  29. Taussky HH, Shorr E: A microcolorimetric method for the determination of inorganic phosphorus. J Biol Chem 202: 675–685, 1953

    PubMed  Google Scholar 

  30. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with folin phenol reagent. J Biol Chem 193: 265–275, 1953

    Google Scholar 

  31. Laemmli UK: Cleavage of structural proteins during the assembly of the heart of the bacteriophage T4. Nature 227: 680–689, 1970

    PubMed  Google Scholar 

  32. Morrissey JH: Silver stain for proteins in polyacrylamide gels: a modified procedure with enhanced uniform sensitivity. Anal Biochem 117: 307–310, 1981

    Google Scholar 

  33. Hirs CHW: Determination of cystine as cysteic acid. Methods Enzymol 11: 59–65, 1967

    Google Scholar 

  34. Matsubara H, Sasaki RM: High recovery of tryptophan from acid hydrolysates of proteins. Biochem Biophys Res Commun 35: 175–181, 1969

    Google Scholar 

  35. Nikodem V, Fresco JR: Protein finger printing by SDS gel electrophoresis after partial fragmentation with CNBr. Anal Biochem 97: 382–386, 1979

    Google Scholar 

  36. Pierce GN, Kutryk MJB, Dhalla NS: Alteration in calcium binding by and composition of the cardiac sarcolemmal membrane in chronic diabetes. Proc Natl Acad Sci USA 80: 5412–5418, 1983

    Google Scholar 

  37. Segrest JP, Jackson RL: Molecular weight determination of glycoproteins by polyacrylamide electrophoresis in sodium docyl sulfate. Methods Enzymol 28: 54–65, 1972

    Google Scholar 

  38. Janda J, Work E: A colorimetric estimation of lipopolysaccharides. FEBS Lett 16: 343–345, 1971

    Google Scholar 

  39. Aminoff D: Methods of the quantitative estimation of Nacetyneuraminic acid and their application to hydrolysates of sialomueoids. Biochem J 81: 384–392, 1961

    Google Scholar 

  40. Brandl CJ, Green NM, Korczak B, MacLennan DH: Two Cau2+ ATPase genes; homologies and mechanistic implications of deduced amino acid sequences. Cell 44: 597–607, 1986

    Google Scholar 

  41. Hewick RM, Hunkapillar MW, Hood LE, Dreyer WJ: A gas-liquid solid phase peptide and protein sequencer. J Biol Chem 265: 7990–7997, 1981

    Google Scholar 

  42. Tada M, Yamamoto T, Tonomura Y: Molecular mechanism of active calcium transport by sarcoplasmic reticulum. Physiol Rev 58: 1–79, 1978

    Google Scholar 

  43. Moulton MP, Sabbadini RA, Norton KC, Dahms AS: Studies on the transverse tubule membrane Mg-ATPase. J Biol Chem 261: 12244–12251, 1986

    Google Scholar 

  44. Langer GA: The structure and function of the myocardial cell surface. Am J Physiol 235: H461-H470, 1978

    Google Scholar 

  45. Philipson KD, Bers DM, Nishimoto AY: The role of phospholipids in the Ca2+ binding of isolated cardiac sarcolemma. J Mol Cell Cardiol 12: 1159–1164, 1980

    Google Scholar 

  46. Caroni P, Zurini M, Clark A, Carafoli E: Further characterization and reconstitution of the purified Ca2+-pumping ATPase of heart sarcolemma. J Biol Chem 258: 7305–7310, 1983

    Google Scholar 

  47. Moffat MP, Dhalla NS: Heart sarcolemmal ATPase and calcium binding activities in rats fed a high cholesterol diet. Can J Cardiol 1: 194–200, 1985

    Google Scholar 

  48. Warren GB, Honslay MD, Metcalf JC, Birdsall NJM: Cholesterol is excluded from the phospholipid annulus surrounding an active calcium transport protein. Nature 255: 684–687, 1975

    Google Scholar 

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Authors and Affiliations

  1. Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, R2H 2A6, Winnipeg, Canada

    Dayuan Zhao & Naranjan S. Dhalla

  2. Department of Physiology, Faculty of Medicine, University of Manitoba, R2H 2A6, Winnipeg, Canada

    Dayuan Zhao & Naranjan S. Dhalla

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  1. Dayuan Zhao
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  2. Naranjan S. Dhalla
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Zhao, D., Dhalla, N.S. Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane. Mol Cell Biochem 107, 135–149 (1991). https://doi.org/10.1007/BF00225517

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  • DOI: https://doi.org/10.1007/BF00225517

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