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Evidence for high molecular weight Na−Ca exchange in cardiac sarcolemmal vesicles

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Summary

Cardiac sarcolemma (SL) vesicles were subjected to irradiation inactivation-target sizing analyses and gel permeation high performance liquid chromatography (HPLC) to ascertain the weight range of native Na−Ca exchange. Frozen SL vesicle preparations were irradiated by electron bombardment and assayed for Na−Ca exchange activity. When applied to classical target sizing theory, the results yielded a minimum molecular weight (M r) of approximately 226,000±20,000sd (n=6). SL vesicle proteins were solubilized in 6% sodium cholate in the presence of exogenous phospholipid and fractionated by size on a TSK 30XL HPLC column. Eluted proteins were mixed 1∶1 with mobile phase buffer containing 50mg/ml soybean phospholipid and reconstituted by detergent dilution. The resulting proteoliposomes were assayed for Na−Ca exchange activity. Na−Ca exchange activity eluted in early fractions containing larger proteins as revealed by SDS-PAGE. Recovery of total protein and Na−Ca exchange activity were 91±7 and 68±11%, respectively. In the peak fraction, Na−Ca exchange specific activity increased two-to threefold compared to reconstituted controls. Compared to the elution profile of protein standards under identical column conditions, sodium cholate solubilized exchange activity had a minimumM r of 224,000 Da. Specific45Ca2+-binding SL proteins withM r of 234,000, 112,000, and 90,000 Da were detected by autoradiography of proteins transferred electrophoretically to nitrocellulose.

These data suggest that native cardiac Na−Ca exchange is approximately 225,000 Da or larger. The exact identification and purification of cardiac Na−Ca exchange protein(s) remains incomplete.

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References

  1. Ahmed, Z., Reis, J.L. 1958. The activation and inhibition of 5′-nucleotidase.Biochem. J. 69:386–387

    PubMed  Google Scholar 

  2. Baron, M.D., Luzio, J.P. 1987. The synthesis and turnover of 5′-nucleotidase in primary cultured hepatocytes.Biochim. Biophys. Acta 927:81–85

    PubMed  Google Scholar 

  3. Barzilai, R., Rahamimoff, H. 1987. Immunological Identification of the synaptic plasma membrane Na+−Ca2+ exchanger.J. Biol. Chem. 262:10315–10320

    Google Scholar 

  4. Barzilai, A., Spanier, R., Rahamimoff, H. 1984. Isolation, purification, and reconstitution of the Na+ gradient-dependent Ca2+ transporter (Na+−Ca2+ exchanger) from brain synaptic membranes.Proc. Natl. Acad. Sci. USA 81:6521–6525

    PubMed  Google Scholar 

  5. Dale, W.E., Tsai, Y.-S., Yung, C.Y., Hale, C.C., Rovetto, M.J., Kim, H.D. 1988. Kinetic characterization and radiation-target sizing of the glucose transporter in cardia sarcolemmal vesicles.Biochim. Biophys. Acta (in press)

  6. Hale, C.C., Slaughter, R.S., Ahrens, D.C., Reeves, J.P. 1984. Identification and partial purification of the cardiac sodium-calcium exchange protein.Proc. Natl. Acad. Sci. USA 81:6569–6573

    PubMed  Google Scholar 

  7. Helenius, A., McCaslin, D.R., Fries, E., Tanford, C. 1979. Properties of detergents.Meth. Enzymol. 56:734–758

    PubMed  Google Scholar 

  8. Jung, C.Y., Hsu, T.L., Hah, J.S., Cha, C., Haas, M.N. 1980. Glucose transport carrier of human erythrocytes.J. Biol. Chem. 255:361–364

    PubMed  Google Scholar 

  9. Kempner, E.S., Miller, J.H., McCreery, M.J. 1986. Radiation target analysis of glycoproteinsAnal. Biochem. 156:140–146

    PubMed  Google Scholar 

  10. Kempner, E.S., Schlegel, W. 1979. Size determination of enzymes by radiation inactivation.Anal. Biochem. 92:2–10

    PubMed  Google Scholar 

  11. Kepner, G.R., Macey, R.I. 1968. Membrane enzyme systems. Molecular size determinations by radiation inactivation.Biochim. Biophys. Acta 163:188–203

    PubMed  Google Scholar 

  12. Kuwayama, H., Kanazawa, T. 1982. Purification of cardiac sarcolemmal vesicles: High sodium pump content and ATP-dependent calmodulin-activated calcium uptake.J. Biochem. 91:1419–1426

    PubMed  Google Scholar 

  13. Laemmli, V.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685

    Google Scholar 

  14. LeMaire, M., Aggerbeck, L.P., Monteilhet, C., Andersen, J.P., Moller, J.V. 1986. The use of high-performance liquid chromatography for the determination of size and molecular weight of proteins: A caution and a list of membrane proteins suitable as standards.Anal. Biochem. 154:525–535

    PubMed  Google Scholar 

  15. Letarte, M., Quakenbush, E.J., Baumal, R., Michalak, M. 1986. Correlations between the 44D7 antigenic complex and the plasma membrane Na+−Ca2+ exchanger.Biochem. Cell Biol. 64:1160–1169

    PubMed  Google Scholar 

  16. Longoni, S., Carafoli, E. 1987. Identification of the Na+/Ca2+ exchanger of calf heart sarcolemma with the help of specific antibodies.Biochem. Biophys. Res. Commun. 145:1059–1063

    PubMed  Google Scholar 

  17. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275

    PubMed  Google Scholar 

  18. Maruyama, K., Mikawa, T., Ebashi, S. 1984. Detection of calcium binding proteins by45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis.J. Biochem. 95:511–519

    PubMed  Google Scholar 

  19. McGrew, S.G., Boucek, R.J., McIntyre, J.O., Jung, C.Y., Fleischer, S. 1987. Target size of the ryanodine receptor from junctional terminal cisternae of sarcoplasmic reticulum.Biochemistry 26:3183–3187

    PubMed  Google Scholar 

  20. Michalak, M., Quakenbush, E.J., Letarte, M. 1986. Inhibition of Na+/Ca2+ exchanger activity in cardiac and skeletal muscle sarcolemmal vesicles by monoclonal antibody 44D7.J. Biol. Chem. 261:92–95

    PubMed  Google Scholar 

  21. Miyamoto, H., Racker, E.F. 1980. Solubilization and partial purification of the Ca2+/Na+ antiporter from the plasma membrane of bovine heart.J. Biol. Chem. 255:2656–2658

    PubMed  Google Scholar 

  22. Newman, M.J., Foster, D.L., Wilson, T.H., Kaback, H.R. 1981. Purification and reconstitution of functional lactose carrier fromEscherichia coli.J. Biol. Chem. 256:11804–11808

    Google Scholar 

  23. Peña, P. de la, Hale, C.C., Reeves, J.P. 1985. The cardiac sarcolemmal Na−Ca exchanger is a glycoprotein.Biophys. J. 47:271a

    Google Scholar 

  24. Philipson, K.D., Nishimoto, A.Y. 1982. Stimulation of Na+−Ca2+ exchanger in cardiac sarcolemmal vesicles by proteinase pretreatment.Am. J. Physiol. 243:C191-C195

    PubMed  Google Scholar 

  25. Reeves, J.R. 1985. The sarcolemmal sodium-calcium exchange system.In: Regulation of Calcium Transport Across Muscle Membranes. A.E. Shamoo, editor. pp. 77–127. Academic, New York

    Google Scholar 

  26. Reeves, J.P., Sutko, J.L. 1979. Sodium-calcium ion exchange in cardiac membrane vesicles.Proc. Natl. Acad. Sci. USA 76:590–594

    PubMed  Google Scholar 

  27. Reeves, J.P., Sutko, J.L. 1983. Competitive interactions of sodium and calcium with the sodium-calcium exchange system of cardiac sarcolemmal vesicles.J. Biol. Chem. 258:3178–3182

    PubMed  Google Scholar 

  28. Reuter, H. 1982. Na−Ca countertransport in cardiac muscle.In: Membranes and Transport. A.N. Martanasi, editor. pp. 623–631. Plenum, New York

    Google Scholar 

  29. Schaffner, W., Weissmann, C. 1973. A rapid, sensitive, and specific method for the determinationof protein in dilute solution.Anal. Biochem. 56:502–514

    Article  PubMed  Google Scholar 

  30. Slaughter, R.S., Sutko, J.L., Reeves, J.P. 1983. Equilibrium calcium-calcium exchange in cardiac sarcolemmal vesicles.J. Biol. Chem. 258:3183–3190

    PubMed  Google Scholar 

  31. Soldati, L., Longoni, S., Carafoli, E. 1985. Solubilization and reconstitution of the Na+/Ca2+ exchanger of cardiac sarcolemma.J. Biol. Chem. 260:13321–13327

    Google Scholar 

  32. Thompson, L.F., Ruedi, J.M., Low, M.G. 1987. Purification of 5′-nucleotidase from human placenta after release from plasma membranes by phosphotidylinositol-specific phospholipase C.Biochem. Biophys. Res. Commun. 145:118–125

    PubMed  Google Scholar 

  33. 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

    PubMed  Google Scholar 

  34. Wakabayashi, S., Goshima, K. 1982. Partial purification of Na+−Ca2+ antiporter from plasma membrane of chick heart.Biochim. Biophys. Acta 693:125–133

    PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Veterinary Biomedical Sciences, University of Missouri-Columbia, 65211, Columbia, Missoúri

    Calvin C. Hale & Steven B. Kleiboeker

  2. Department of The John M. Dalton Research Center, University of Missouri-Columbia, 65211, Columbia, Missoúri

    Calvin C. Hale, Steven B. Kleiboeker, Carol G. Carlton & Michael J. Rovetto

  3. Department of Physiology, University of Missouri-Columbia, 65211, Columbia, Missoúri

    Michael J. Rovetto

  4. Department of Pharmacology, University of Missouri-Columbia, 65211, Columbia, Missoúri

    Chan Jung

  5. Veterans Administration Hospital, 14215, Buffalo, New York

    H. D. Kim

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Hale, C.C., Kleiboeker, S.B., Carlton, C.G. et al. Evidence for high molecular weight Na−Ca exchange in cardiac sarcolemmal vesicles. J. Membrain Biol. 106, 211–218 (1988). https://doi.org/10.1007/BF01872159

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  • DOI: https://doi.org/10.1007/BF01872159

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