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. 2000 Mar;78(3):1306–1323. doi: 10.1016/S0006-3495(00)76686-0

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells: new insights on Ca-ATPase regulation by phospholamban.

J E Mahaney 1, J M Autry 1, L R Jones 1
PMCID: PMC1300731  PMID: 10692318

Abstract

Kinetics studies of the cardiac Ca-ATPase expressed in Sf21 cells (Spodoptera frugiperda insect cells) have been carried out to test the hypotheses that phospholamban inhibits Ca-ATPase cycling by decreasing the rate of the E1.Ca to E1'.Ca transition and/or the rate of phosphoenzyme hydrolysis. Three sample types were studied: Ca-ATPase expressed alone, Ca-ATPase coexpressed with wild-type phospholamban (the natural pentameric inhibitor), and Ca-ATPase coexpressed with the L37A-phospholamban mutant (a more potent monomeric inhibitor, in which Leu(37) is replaced by Ala). Phospholamban coupling to the Ca-ATPase was controlled using a monoclonal antibody against phospholamban. Gel electrophoresis and immunoblotting confirmed an equivalent ratio of Ca-ATPase and phospholamban in each sample (1 mol Ca-ATPase to 1.5 mol phospholamban). Steady-state ATPase activity assays at 37 degrees C, using 5 mM MgATP, showed that the phospholamban-containing samples had nearly equivalent maximum activity ( approximately 0.75 micromol. nmol Ca-ATPase(-1).min(-1) at 15 microM Ca(2+)), but that wild-type phospholamban and L37A-phospholamban increased the Ca-ATPase K(Ca) values by 200 nM and 400 nM, respectively. When steady-state Ca-ATPase phosphoenzyme levels were measured at 0 degrees C, using 1 microM MgATP, the K(Ca) values also shifted by 200 nM and 400 nM, respectively, similar to the results obtained by measuring ATP hydrolysis at 37 degrees C. Measurements of the time course of phosphoenzyme formation at 0 degrees C, using 1 microM MgATP and 268 nM ionized [Ca(2+)], indicated that L37A-phospholamban decreased the steady-state phosphoenzyme level to a greater extent (45%) than did wild-type phospholamban (33%), but neither wild-type nor L37A-phospholamban had any effect on the apparent rate of phosphoenzyme formation relative to that of Ca-ATPase expressed alone. Measurements of inorganic phosphate (P(i)) release concomitant with the phosphoenzyme formation studies showed that L37A-phospholamban decreased the steady-state rate of P(i) release to a greater extent (45%) than did wild-type phospholamban (33%). However, independent measurements of Ca-ATPase dephosphorylation after the addition of 5 mM EGTA to the phosphorylated enzyme showed that neither wild-type phospholamban nor L37A-phospholamban had any effect on the rate of phosphoenzyme decay relative to Ca-ATPase expressed alone. Computer simulation of the kinetics data indicated that phospholamban and L37A-phospholamban decreased twofold and fourfold, respectively, the equilibrium binding of the first Ca(2+) ion to the Ca-ATPase E1 intermediate, rather than inhibiting rate of the E.Ca to E'.Ca transition or the rate of phosphoenzyme decay. Therefore, we conclude that phospholamban inhibits Ca-ATPase cycling by decreasing Ca-ATPase Ca(2+) binding to the E1 intermediate.

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Selected References

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  1. Antipenko A. Y., Spielman A. I., Kirchberger M. A. Comparison of the effects of phospholamban and jasmone on the calcium pump of cardiac sarcoplasmic reticulum. Evidence for modulation by phospholamban of both Ca2+ affinity and Vmax (Ca) of calcium transport. J Biol Chem. 1997 Jan 31;272(5):2852–2860. doi: 10.1074/jbc.272.5.2852. [DOI] [PubMed] [Google Scholar]
  2. Antipenko A. Y., Spielman A. I., Sassaroli M., Kirchberger M. A. Comparison of the kinetic effects of phospholamban phosphorylation and anti-phospholamban monoclonal antibody on the calcium pump in purified cardiac sarcoplasmic reticulum membranes. Biochemistry. 1997 Oct 21;36(42):12903–12910. doi: 10.1021/bi971109v. [DOI] [PubMed] [Google Scholar]
  3. Antipenko A., Spielman A. I., Kirchberger M. A. Kinetic differences in the phospholamban-regulated calcium pump when studied in crude and purified cardiac sarcoplasmic reticulum vesicles. J Membr Biol. 1999 Feb 1;167(3):257–265. doi: 10.1007/s002329900490. [DOI] [PubMed] [Google Scholar]
  4. Autry J. M., Jones L. R. Functional Co-expression of the canine cardiac Ca2+ pump and phospholamban in Spodoptera frugiperda (Sf21) cells reveals new insights on ATPase regulation. J Biol Chem. 1997 Jun 20;272(25):15872–15880. doi: 10.1074/jbc.272.25.15872. [DOI] [PubMed] [Google Scholar]
  5. Autry J. M., Jones L. R. High-level coexpression of the canine cardiac calcium pump and phospholamban in Sf21 insect cells. Ann N Y Acad Sci. 1998 Sep 16;853:92–102. doi: 10.1111/j.1749-6632.1998.tb08259.x. [DOI] [PubMed] [Google Scholar]
  6. Barshop B. A., Wrenn R. F., Frieden C. Analysis of numerical methods for computer simulation of kinetic processes: development of KINSIM--a flexible, portable system. Anal Biochem. 1983 Apr 1;130(1):134–145. doi: 10.1016/0003-2697(83)90660-7. [DOI] [PubMed] [Google Scholar]
  7. Briggs F. N., Lee K. F., Wechsler A. W., Jones L. R. Phospholamban expressed in slow-twitch and chronically stimulated fast-twitch muscles minimally affects calcium affinity of sarcoplasmic reticulum Ca(2+)-ATPase. J Biol Chem. 1992 Dec 25;267(36):26056–26061. [PubMed] [Google Scholar]
  8. Cantilina T., Sagara Y., Inesi G., Jones L. R. Comparative studies of cardiac and skeletal sarcoplasmic reticulum ATPases. Effect of a phospholamban antibody on enzyme activation by Ca2+. J Biol Chem. 1993 Aug 15;268(23):17018–17025. [PubMed] [Google Scholar]
  9. Chu G., Li L., Sato Y., Harrer J. M., Kadambi V. J., Hoit B. D., Bers D. M., Kranias E. G. Pentameric assembly of phospholamban facilitates inhibition of cardiac function in vivo. J Biol Chem. 1998 Dec 11;273(50):33674–33680. doi: 10.1074/jbc.273.50.33674. [DOI] [PubMed] [Google Scholar]
  10. Coll K. E., Johnson R. G., Jr, McKenna E. Relationship between phospholamban and nucleotide activation of cardiac sarcoplasmic reticulum Ca2+ adenosinetriphosphatase. Biochemistry. 1999 Feb 23;38(8):2444–2451. doi: 10.1021/bi9823028. [DOI] [PubMed] [Google Scholar]
  11. Froehlich J. P., Taylor E. W. Transient state kinetic effects of calcium ion on sarcoplasmic reticulum adenosine triphosphatase. J Biol Chem. 1976 Apr 25;251(8):2307–2315. [PubMed] [Google Scholar]
  12. Froehlich J. P., Taylor E. W. Transient state kinetic studies of sarcoplasmic reticulum adenosine triphosphatase. J Biol Chem. 1975 Mar 25;250(6):2013–2021. [PubMed] [Google Scholar]
  13. 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]
  14. Harrison S. M., Bers D. M. Correction of proton and Ca association constants of EGTA for temperature and ionic strength. Am J Physiol. 1989 Jun;256(6 Pt 1):C1250–C1256. doi: 10.1152/ajpcell.1989.256.6.C1250. [DOI] [PubMed] [Google Scholar]
  15. Hughes G., East J. M., Lee A. G. The hydrophilic domain of phospholamban inhibits the Ca2+ transport step of the Ca(2+)-ATPase. Biochem J. 1994 Oct 15;303(Pt 2):511–516. doi: 10.1042/bj3030511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Inesi G., Kurzmack M., Coan C., Lewis D. E. Cooperative calcium binding and ATPase activation in sarcoplasmic reticulum vesicles. J Biol Chem. 1980 Apr 10;255(7):3025–3031. [PubMed] [Google Scholar]
  17. 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]
  18. Jones L. R., Besch H. R., Jr, Watanabe A. M. Regulation of the calcium pump of cardiac sarcoplasmic reticulum. Interactive roles of potassium and ATP on the phosphoprotein intermediate of the (K+,Ca2+)-ATPase. J Biol Chem. 1978 Mar 10;253(5):1643–1653. [PubMed] [Google Scholar]
  19. 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]
  20. Jones L. R., Phan S. H., Besch H. R., Jr Gel electrophoretic and density gradient analysis of the (K+ + Ca2+)-ATPase and the (Na+ + K+)-ATPase activities of cardiac membrane vesicles. Biochim Biophys Acta. 1978 Dec 19;514(2):294–309. doi: 10.1016/0005-2736(78)90300-0. [DOI] [PubMed] [Google Scholar]
  21. Kimura Y., Kurzydlowski K., Tada M., MacLennan D. H. Phospholamban inhibitory function is activated by depolymerization. J Biol Chem. 1997 Jun 13;272(24):15061–15064. doi: 10.1074/jbc.272.24.15061. [DOI] [PubMed] [Google Scholar]
  22. Kimura Y., Kurzydlowski K., Tada M., MacLennan D. H. Phospholamban regulates the Ca2+-ATPase through intramembrane interactions. J Biol Chem. 1996 Sep 6;271(36):21726–21731. doi: 10.1074/jbc.271.36.21726. [DOI] [PubMed] [Google Scholar]
  23. Kranias E. G., Mandel F., Wang T., Schwartz A. Mechanism of the stimulation of calcium ion dependent adenosine triphosphatase of cardiac sarcoplasmic reticulum by adenosine 3',5'-monophosphate dependent protein kinase. Biochemistry. 1980 Nov 11;19(23):5434–5439. doi: 10.1021/bi00564a044. [DOI] [PubMed] [Google Scholar]
  24. Kranias E. G. Regulation of Ca2+ transport by cyclic 3',5'-AMP-dependent and calcium-calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum. Biochim Biophys Acta. 1985 Feb 21;844(2):193–199. doi: 10.1016/0167-4889(85)90090-4. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Lanzetta P. A., Alvarez L. J., Reinach P. S., Candia O. A. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979 Nov 15;100(1):95–97. doi: 10.1016/0003-2697(79)90115-5. [DOI] [PubMed] [Google Scholar]
  27. MacLennan D. H., Toyofuku T., Lytton J. Structure-function relationships in sarcoplasmic or endoplasmic reticulum type Ca2+ pumps. Ann N Y Acad Sci. 1992 Nov 30;671:1–10. doi: 10.1111/j.1749-6632.1992.tb43779.x. [DOI] [PubMed] [Google Scholar]
  28. Mahaney J. E., Froehlich J. P., Thomas D. D. Conformational transitions of the sarcoplasmic reticulum Ca-ATPase studied by time-resolved EPR and quenched-flow kinetics. Biochemistry. 1995 Apr 11;34(14):4864–4879. doi: 10.1021/bi00014a044. [DOI] [PubMed] [Google Scholar]
  29. McKenna E., Smith J. S., Coll K. E., Mazack E. K., Mayer E. J., Antanavage J., Wiedmann R. T., Johnson R. G., Jr Dissociation of phospholamban regulation of cardiac sarcoplasmic reticulum Ca2+ATPase by quercetin. J Biol Chem. 1996 Oct 4;271(40):24517–24525. doi: 10.1074/jbc.271.40.24517. [DOI] [PubMed] [Google Scholar]
  30. Movsesian M. A., Karimi M., Green K., Jones L. R. Ca(2+)-transporting ATPase, phospholamban, and calsequestrin levels in nonfailing and failing human myocardium. Circulation. 1994 Aug;90(2):653–657. doi: 10.1161/01.cir.90.2.653. [DOI] [PubMed] [Google Scholar]
  31. Negash S., Chen L. T., Bigelow D. J., Squier T. C. Phosphorylation of phospholamban by cAMP-dependent protein kinase enhances interactions between Ca-ATPase polypeptide chains in cardiac sarcoplasmic reticulum membranes. Biochemistry. 1996 Sep 3;35(35):11247–11259. doi: 10.1021/bi960864q. [DOI] [PubMed] [Google Scholar]
  32. Porzio M. A., Pearson A. M. Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochim Biophys Acta. 1977 Jan 25;490(1):27–34. doi: 10.1016/0005-2795(77)90102-7. [DOI] [PubMed] [Google Scholar]
  33. Reddy L. G., Jones L. R., Pace R. C., Stokes D. L. Purified, reconstituted cardiac Ca2+-ATPase is regulated by phospholamban but not by direct phosphorylation with Ca2+/calmodulin-dependent protein kinase. J Biol Chem. 1996 Jun 21;271(25):14964–14970. doi: 10.1074/jbc.271.25.14964. [DOI] [PubMed] [Google Scholar]
  34. Sham J. S., Jones L. R., Morad M. Phospholamban mediates the beta-adrenergic-enhanced Ca2+ uptake in mammalian ventricular myocytes. Am J Physiol. 1991 Oct;261(4 Pt 2):H1344–H1349. doi: 10.1152/ajpheart.1991.261.4.H1344. [DOI] [PubMed] [Google Scholar]
  35. Shigekawa M., Finegan J. A., Katz A. M. Calcium transport ATPase of canine cardiac sarcoplasmic reticulum. A comparison with that of rabbit fast skeletal muscle sarcoplasmic reticulum. J Biol Chem. 1976 Nov 25;251(22):6894–6900. [PubMed] [Google Scholar]
  36. Simmerman H. K., Jones L. R. Phospholamban: protein structure, mechanism of action, and role in cardiac function. Physiol Rev. 1998 Oct;78(4):921–947. doi: 10.1152/physrev.1998.78.4.921. [DOI] [PubMed] [Google Scholar]
  37. Simmerman H. K., Kobayashi Y. M., Autry J. M., Jones L. R. A leucine zipper stabilizes the pentameric membrane domain of phospholamban and forms a coiled-coil pore structure. J Biol Chem. 1996 Mar 8;271(10):5941–5946. doi: 10.1074/jbc.271.10.5941. [DOI] [PubMed] [Google Scholar]
  38. Smith P. D., Liesegang G. W., Berger R. L., Czerlinski G., Podolsky R. J. A stopped-flow investigation of calcium ion binding by ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid. Anal Biochem. 1984 Nov 15;143(1):188–195. doi: 10.1016/0003-2697(84)90575-x. [DOI] [PubMed] [Google Scholar]
  39. Sumida M., Wang T., Mandel F., Froehlich J. P., Schwartz A. Transient kinetics of Ca2+ transport of sarcoplasmic reticulum. A comparison of cardiac and skeletal muscle. J Biol Chem. 1978 Dec 25;253(24):8772–8777. [PubMed] [Google Scholar]
  40. Sumida M., Wang T., Schwartz A., Younkin C., Froehlich J. P. The Ca2+-ATPase partial reactions in cardiac and skeletal sarcoplasmic reticulum. A comparison of transient state kinetic data. J Biol Chem. 1980 Feb 25;255(4):1497–1503. [PubMed] [Google Scholar]
  41. Tada M., Ohmori F., Yamada M., Abe H. Mechanism of the stimulation of Ca2+-dependent ATPase of cardiac sarcoplasmic reticulum by adenosine 3':5'-monophosphate-dependent protein kinase. Role of the 22,000-dalton protein. J Biol Chem. 1979 Jan 25;254(2):319–326. [PubMed] [Google Scholar]
  42. Tada M., Yamada M., Ohmori F., Kuzuya T., Inui M., Abe H. Transient state kinetic studies of Ca2+-dependent ATPase and calcium transport by cardiac sarcoplasmic reticulum. Effect of cyclic AMP-dependent protein kinase-catalyzed phosphorylation of phospholamban. J Biol Chem. 1980 Mar 10;255(5):1985–1992. [PubMed] [Google Scholar]
  43. Toyofuku T., Kurzydlowski K., Tada M., MacLennan D. H. Amino acids Glu2 to Ile18 in the cytoplasmic domain of phospholamban are essential for functional association with the Ca(2+)-ATPase of sarcoplasmic reticulum. J Biol Chem. 1994 Jan 28;269(4):3088–3094. [PubMed] [Google Scholar]
  44. Toyofuku T., Kurzydlowski K., Tada M., MacLennan D. H. Amino acids Lys-Asp-Asp-Lys-Pro-Val402 in the Ca(2+)-ATPase of cardiac sarcoplasmic reticulum are critical for functional association with phospholamban. J Biol Chem. 1994 Sep 16;269(37):22929–22932. [PubMed] [Google Scholar]
  45. 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]

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