Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 1995 May;68(5):1787–1795. doi: 10.1016/S0006-3495(95)80355-3

Molecular dynamics in mouse atrial tumor sarcoplasmic reticulum.

J C Voss 1, J E Mahaney 1, L R Jones 1, D D Thomas 1
PMCID: PMC1282081  PMID: 7612820

Abstract

We have determined directly the effects of the inhibitory peptide phospholamban (PLB) on the rotational dynamics of the calcium pump (Ca-ATPase) of cardiac sarcoplasmic reticulum (SR). This was accomplished by comparing mouse ventricular SR, which has PLB levels similar to those found in other mammals, with mouse atrial SR, which is effectively devoid of PLB and thus has much higher (unregulated) calcium pump activity. To obtain sufficient quantities of atrial SR, we isolated the membranes from atrial tumor cells. We used time-resolved phosphorescence anisotropy of an erythrosin isothiocyanate label attached selectively and rigidly to the Ca-ATPase, to detect the microsecond rotational motion of the Ca-ATPase in the two preparations. The time-resolved phosphorescence anisotropy decays of both preparations at 25 degrees C were multi-exponential, because of the presence of different oligomeric species. The rotational correlation times for the different oligomers were similar for the two preparations, but the total decay amplitude was substantially greater for atrial tumor SR, indicating that a smaller fraction of the Ca-ATPase molecules exists as large aggregates. Phosphorylation of PLB in ventricular SR decreased the population of large-scale Ca-ATPase aggregates to a level similar to that of atrial tumor SR. Lipid chain mobility (fluidity), detected by electron paramagnetic resonance of stearic acid spin labels, was very similar in the two preparations, indicating that the higher protein mobility in atrial tumor SR is not due to higher lipid fluidity. We conclude that PLB inhibits by inducing Ca-ATPase lateral aggregation, which can be relieved either by phosphorylating or removing PLB.

Full text

PDF
1787

Images in this article

1789FIGURE 1
on p.1789
1789FIGURE 2
on p.1789

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bigelow D. J., Thomas D. D. Rotational dynamics of lipid and the Ca-ATPase in sarcoplasmic reticulum. The molecular basis of activation by diethyl ether. J Biol Chem. 1987 Oct 5;262(28):13449–13456. [PubMed] [Google Scholar]
  2. Birmachu W., Thomas D. D. Rotational dynamics of the Ca-ATPase in sarcoplasmic reticulum studied by time-resolved phosphorescence anisotropy. Biochemistry. 1990 Apr 24;29(16):3904–3914. doi: 10.1021/bi00468a017. [DOI] [PubMed] [Google Scholar]
  3. Birmachu W., Voss J. C., Louis C. F., Thomas D. D. Protein and lipid rotational dynamics in cardiac and skeletal sarcoplasmic reticulum detected by EPR and phosphorescence anisotropy. Biochemistry. 1993 Sep 14;32(36):9445–9453. doi: 10.1021/bi00087a024. [DOI] [PubMed] [Google Scholar]
  4. Cherry R. J. Measurement of protein rotational diffusion in membranes by flash photolysis. Methods Enzymol. 1978;54:47–61. doi: 10.1016/s0076-6879(78)54007-x. [DOI] [PubMed] [Google Scholar]
  5. Chiesi M., Schwaller R. Involvement of electrostatic phenomena in phospholamban-induced stimulation of Ca uptake into cardiac sarcoplasmic reticulum. FEBS Lett. 1989 Feb 13;244(1):241–244. doi: 10.1016/0014-5793(89)81201-3. [DOI] [PubMed] [Google Scholar]
  6. Davis B. A., Schwartz A., Samaha F. J., Kranias E. G. Regulation of cardiac sarcoplasmic reticulum calcium transport by calcium-calmodulin-dependent phosphorylation. J Biol Chem. 1983 Nov 25;258(22):13587–13591. [PubMed] [Google Scholar]
  7. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378–417. doi: 10.1016/0076-6879(88)57093-3. [DOI] [PubMed] [Google Scholar]
  8. Field L. J. Atrial natriuretic factor-SV40 T antigen transgenes produce tumors and cardiac arrhythmias in mice. Science. 1988 Feb 26;239(4843):1029–1033. doi: 10.1126/science.2964082. [DOI] [PubMed] [Google Scholar]
  9. Fujii J., Maruyama K., Tada M., MacLennan D. H. Co-expression of slow-twitch/cardiac muscle Ca2(+)-ATPase (SERCA2) and phospholamban. FEBS Lett. 1990 Oct 29;273(1-2):232–234. doi: 10.1016/0014-5793(90)81092-3. [DOI] [PubMed] [Google Scholar]
  10. Fujii J., Ueno A., Kitano K., Tanaka S., Kadoma M., Tada M. Complete complementary DNA-derived amino acid sequence of canine cardiac phospholamban. J Clin Invest. 1987 Jan;79(1):301–304. doi: 10.1172/JCI112799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Inesi G. Mechanism of calcium transport. Annu Rev Physiol. 1985;47:573–601. doi: 10.1146/annurev.ph.47.030185.003041. [DOI] [PubMed] [Google Scholar]
  12. Inui M., Chamberlain B. K., Saito A., Fleischer S. The nature of the modulation of Ca2+ transport as studied by reconstitution of cardiac sarcoplasmic reticulum. J Biol Chem. 1986 Feb 5;261(4):1794–1800. [PubMed] [Google Scholar]
  13. Jones L. R., Cala S. E. Biochemical evidence for functional heterogeneity of cardiac sarcoplasmic reticulum vesicles. J Biol Chem. 1981 Nov 25;256(22):11809–11818. [PubMed] [Google Scholar]
  14. Jones L. R., Field L. J. Residues 2-25 of phospholamban are insufficient to inhibit Ca2+ transport ATPase of cardiac sarcoplasmic reticulum. J Biol Chem. 1993 Jun 5;268(16):11486–11488. [PubMed] [Google Scholar]
  15. Jones L. R., Simmerman H. K., Wilson W. W., Gurd F. R., Wegener A. D. Purification and characterization of phospholamban from canine cardiac sarcoplasmic reticulum. J Biol Chem. 1985 Jun 25;260(12):7721–7730. [PubMed] [Google Scholar]
  16. Jorgensen A. O., Jones L. R. Localization of phospholamban in slow but not fast canine skeletal muscle fibers. An immunocytochemical and biochemical study. J Biol Chem. 1986 Mar 15;261(8):3775–3781. [PubMed] [Google Scholar]
  17. Karon B. S., Thomas D. D. Molecular mechanism of Ca-ATPase activation by halothane in sarcoplasmic reticulum. Biochemistry. 1993 Jul 27;32(29):7503–7511. doi: 10.1021/bi00080a023. [DOI] [PubMed] [Google Scholar]
  18. Kirchberger M. A., Borchman D., Kasinathan C. Proteolytic activation of the canine cardiac sarcoplasmic reticulum calcium pump. Biochemistry. 1986 Sep 23;25(19):5484–5492. doi: 10.1021/bi00367a021. [DOI] [PubMed] [Google Scholar]
  19. Kirchberger M. A., Tada M. Effects of adenosine 3':5'-monophosphate-dependent protein kinase on sarcoplasmic reticulum isolated from cardiac and slow and fast contracting skeletal muscles. J Biol Chem. 1976 Feb 10;251(3):725–729. [PubMed] [Google Scholar]
  20. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  21. Lipari G., Szabo A. Effect of librational motion on fluorescence depolarization and nuclear magnetic resonance relaxation in macromolecules and membranes. Biophys J. 1980 Jun;30(3):489–506. doi: 10.1016/S0006-3495(80)85109-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ludescher R. D., Thomas D. D. Microsecond rotational dynamics of phosphorescent-labeled muscle cross-bridges. Biochemistry. 1988 May 3;27(9):3343–3351. doi: 10.1021/bi00409a034. [DOI] [PubMed] [Google Scholar]
  23. Mahaney J. E., Kleinschmidt J., Marsh D., Thomas D. D. Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes. Biophys J. 1992 Dec;63(6):1513–1522. doi: 10.1016/S0006-3495(92)81736-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mahaney J. E., Thomas D. D. Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: electron paramagnetic resonance. Biochemistry. 1991 Jul 23;30(29):7171–7180. doi: 10.1021/bi00243a019. [DOI] [PubMed] [Google Scholar]
  25. Manalan A. S., Jones L. R. Characterization of the intrinsic cAMP-dependent protein kinase activity and endogenous substrates in highly purified cardiac sarcolemmal vesicles. J Biol Chem. 1982 Sep 10;257(17):10052–10062. [PubMed] [Google Scholar]
  26. Mersol J. V., Kutchai H., Mahaney J. E., Thomas D. D. Self-association accompanies inhibition of Ca-ATPase by thapsigargin. Biophys J. 1995 Jan;68(1):208–215. doi: 10.1016/S0006-3495(95)80176-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mitchinson C., Wilderspin A. F., Trinnaman B. J., Green N. M. Identification of a labelled peptide after stoicheiometric reaction of fluorescein isothiocyanate with the Ca2+ -dependent adenosine triphosphatase of sarcoplasmic reticulum. FEBS Lett. 1982 Sep 6;146(1):87–92. doi: 10.1016/0014-5793(82)80710-2. [DOI] [PubMed] [Google Scholar]
  28. Simmerman H. K., Collins J. H., Theibert J. L., Wegener A. D., Jones L. R. Sequence analysis of phospholamban. Identification of phosphorylation sites and two major structural domains. J Biol Chem. 1986 Oct 5;261(28):13333–13341. [PubMed] [Google Scholar]
  29. Squier T. C., Bigelow D. J., Thomas D. D. Lipid fluidity directly modulates the overall protein rotational mobility of the Ca-ATPase in sarcoplasmic reticulum. J Biol Chem. 1988 Jul 5;263(19):9178–9186. [PubMed] [Google Scholar]
  30. Squier T. C., Hughes S. E., Thomas D. D. Rotational dynamics and protein-protein interactions in the Ca-ATPase mechanism. J Biol Chem. 1988 Jul 5;263(19):9162–9170. [PubMed] [Google Scholar]
  31. Squier T. C., Thomas D. D. Methodology for increased precision in saturation transfer electron paramagnetic resonance studies of rotational dynamics. Biophys J. 1986 Apr;49(4):921–935. doi: 10.1016/S0006-3495(86)83720-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Squier T. C., Thomas D. D. Relationship between protein rotational dynamics and phosphoenzyme decomposition in the sarcoplasmic reticulum Ca-ATPase. J Biol Chem. 1988 Jul 5;263(19):9171–9177. [PubMed] [Google Scholar]
  33. Squier T. C., Thomas D. D. Selective detection of the rotational dynamics of the protein-associated lipid hydrocarbon chains in sarcoplasmic reticulum membranes. Biophys J. 1989 Oct;56(4):735–748. doi: 10.1016/S0006-3495(89)82721-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tada M., Kadoma M., Fujii J., Kimura Y., Kijima Y. Molecular structure and function of phospholamban: the regulatory protein of calcium pump in cardiac sarcoplasmic reticulum. Adv Exp Med Biol. 1989;255:79–89. doi: 10.1007/978-1-4684-5679-0_9. [DOI] [PubMed] [Google Scholar]
  35. Tada M., Kadoma M., Inui M., Fujii J. Regulation of Ca2+-pump from cardiac sarcoplasmic reticulum. Methods Enzymol. 1988;157:107–154. doi: 10.1016/0076-6879(88)57073-8. [DOI] [PubMed] [Google Scholar]
  36. Tada M., Katz A. M. Phosphorylation of the sarcoplasmic reticulum and sarcolemma. Annu Rev Physiol. 1982;44:401–423. doi: 10.1146/annurev.ph.44.030182.002153. [DOI] [PubMed] [Google Scholar]
  37. Teruel J. A., Inesi G. Roles of phosphorylation and nucleotide binding domains in calcium transport by sarcoplasmic reticulum adenosinetriphosphatase. Biochemistry. 1988 Aug 9;27(16):5885–5890. doi: 10.1021/bi00416a010. [DOI] [PubMed] [Google Scholar]
  38. Toyofuku T., Kurzydlowski K., Tada M., MacLennan D. H. Identification of regions in the Ca(2+)-ATPase of sarcoplasmic reticulum that affect functional association with phospholamban. J Biol Chem. 1993 Feb 5;268(4):2809–2815. [PubMed] [Google Scholar]
  39. Voss J., Birmachu W., Hussey D. M., Thomas D. D. Effects of melittin on molecular dynamics and Ca-ATPase activity in sarcoplasmic reticulum membranes: time-resolved optical anisotropy. Biochemistry. 1991 Jul 30;30(30):7498–7506. doi: 10.1021/bi00244a019. [DOI] [PubMed] [Google Scholar]
  40. Voss J., Jones L. R., Thomas D. D. The physical mechanism of calcium pump regulation in the heart. Biophys J. 1994 Jul;67(1):190–196. doi: 10.1016/S0006-3495(94)80469-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wegener A. D., Simmerman H. K., Liepnieks J., Jones L. R. Proteolytic cleavage of phospholamban purified from canine cardiac sarcoplasmic reticulum vesicles. Generation of a low resolution model of phospholamban structure. J Biol Chem. 1986 Apr 15;261(11):5154–5159. [PubMed] [Google Scholar]
  42. Xu Z. C., Kirchberger M. A. Modulation by polyelectrolytes of canine cardiac microsomal calcium uptake and the possible relationship to phospholamban. J Biol Chem. 1989 Oct 5;264(28):16644–16651. [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES