The Journal of Membrane Biology

, Volume 100, Issue 1, pp 193–205 | Cite as

Trypsin-induced calcium efflux from sarcoplasmic reticulum: Evidence for the involvement of the (Ca2++Mg2+)-ATPase

  • Jun-Ling Huang
  • Traci B. Topping
  • Zhaoping He
  • Brian Folsom
  • A. Keith Dunker


Trypsin digestion of the sarcoplasmic reticulum membrane at 35 to 43°C leads to an increased calcium permeability, the temperature dependence of which suggests tryptic exposure or creation of a channel rather than tryptic release of a mobile carrier (K.C. Toogood et al.,Membr. Biochem.5:49–75, 1983). Here we show that: (1) the digested vesicles both pump and leak calcium, demonstrating that the vesicles remain intact; (2) an increased rate of efflux is not observed for membranes digested and kept at 15°C, but a temperature shift to 35°C following arrested digestion leads to the development of increased calcium permeability, indicating that a digestion step at the lower temperature potentiates increased permeability which develops rapidly as a result of a trypsin-facilitated protein conformational change at the higher temperature; (3) two inhibitors of the ATPase, adenyl-5′-yl imidodiphosphate and dicyclohexyl-carbodiimide, both measurably retard the development of increased permeability at the higher temperature following arrested digestion, suggesting that these inhibitors bind to the target protein and prevent the conformational change responsible for the permeability increase, and further suggesting that the ATPase is the target for the trypsin; (4) digestion of the ATPase at 15°C follows the same initial cleavage pattern as at 35°C, but the cleavage stops or drastically slows down after the second digestion step at the lower temperature, whereas the digestion continues beyond the second step at the higher temperature, showing that an early digestion step may be responsible for potentiating increased permeability; (5) the permeability increase following digestion at 15°C and incubation at 35°C correlates (r>0.98) with the second tryptic cleavage step of the calcium ATPase, providing more support for the ATPase as the trypsin-sensitive efflux site; and (6) the rate of efflux depends on the concentration of the doubly cleaved ATPase molecules to the first power; the null hypothesis that the efflux actually depends on the cleaved ATPase concentration to the second or higher power was examined using the F test and can be rejected (confidence>0.90 to 0.98), suggesting that the efflux pathway is through a single ATPase molecule. We speculate that the pathway for increased calcium permeability is the one employed during calcium uptake and that there is a functional separation of the ATPase and calcium channel activities by trypsin digestion at 15°C followed by incubation at 35°C.

Key Words

sarcoplasmic reticulum trypsin digestion calcium ATPase 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abramson, J.J., Trimm, J.L., Weden, L., Salama, G. 1983. Heavy metals induce rapid calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle.Proc. Natl. Acad. Sci. USA 80:1526–1530PubMedGoogle Scholar
  2. Andersen, J.P., Lassen, K., Møller, J.V. 1985. Changes in Ca2+ affinity related to conformational transitions in the phosphorylated state of soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum.J. Biol. Chem. 260:371–380PubMedGoogle Scholar
  3. Andersen, J.P., Jørgensen, P.L. 1985. Conformational states of sarcoplasmic reticulum Ca2+-ATPase as studied by proteolytic cleavage.J. Membrane Biol. 88:187–198Google Scholar
  4. Andersen, J.P., Møller, J.V., Jørgensen, P.L. 1982. The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements.J. Biol. Chem. 257:8300–8307PubMedGoogle Scholar
  5. Andersen, J.P., Vilsen, B., Collins, J.H., Jørgensen, P.L. 1986. Localization of E1-E2 conformational transitions of sarcoplasmic reticulum Ca2+-ATPase by tryptic cleavage and hydrophobic labeling.J. Membrane Biol. 93:85–92Google Scholar
  6. Berman, M.C. 1982. Energy coupling and uncoupling of active calcium transport by sarcoplasmic reticulum membranes.Biochim. Biophys. Acta 694:95–121PubMedGoogle Scholar
  7. Bhattaharyya, G.K., Johnson, R.A. 1977. Statistical Concepts and Methods. John Wiley & Son, New YorkGoogle Scholar
  8. Bindoli, A., Fleischer, S. 1983. Induced Ca2+ release in skeletal muscle sarcoplasmic reticulum by sulfhydryl reagents and chlorpromazine.Arch. Biochem. Biophys. 221:458–466PubMedGoogle Scholar
  9. Boheim, G., Hanke, W., Eibl, H. 1980. Lipid phase-transition in planar bilayer membrane and its effect on carrier- and poremediated ion-transport.Proc. Natl. Acad. Sci. USA 77:3403–3407PubMedGoogle Scholar
  10. Bradford, M.M. 1976. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 72:248–254PubMedGoogle Scholar
  11. Carvalho, M.D.G.C., Sourza, D. de, Meis, L. de 1976. On a possible mechanism of energy conservation in sarcoplasmic reticulum membrane.J. Biol. Chem. 251:3629–3636PubMedGoogle Scholar
  12. Chen, P.S., Toribara, T.Y., Warner, H. 1956. Microdetermination of phosphorus.Anal. Chem. 28:1756–1758Google Scholar
  13. Chiesi, M. 1984. Cross-linking agents induce rapid calcium release from skeletal muscle sarcoplasmic reticulum.Biochemistry 23:3899–3907PubMedGoogle Scholar
  14. Dunker, A.K., Rueckert, R.R. 1969. Observations on molecular weight determinations on polyacrylamide gel.J. Biol. Chem. 244:5074–5080PubMedGoogle Scholar
  15. Dupont, Y. 1976. Fluorescence studies of the sarcoplasmic reticulum calcium pump.Biochem. Biophys. Res. Commun. 71:544–550PubMedGoogle Scholar
  16. Dux, L., Martonosi, A. 1983. Ca2+-ATPase membrane crystals in sarcoplasmic reticulum. The effect of trypsin digestion.J. Biol. Chem. 258:10111–10115PubMedGoogle Scholar
  17. Ebashi, S., Kodama, A. 1965. A new protein factor promoting aggregation of tropomyosin.J. Biochem. 58:107–108PubMedGoogle Scholar
  18. Folsom, B.F. 1984. Evidence for a Gated Channel Controlling Calcium Efflux from Sarcoplasmic Reticulum. Ph.D. Thesis, Washington State University, Pullman, Wash.Google Scholar
  19. Gingold, M.P., Rigaud, J.L., Champeil, P. 1981. Fluorescence energy transfer between ATPase monomers in sarcoplasmic reticulum reconstituted vesicles.Biochimie 63:923–925PubMedGoogle Scholar
  20. Gould, G.W., Colyer, J., East, J.M., Lee, A.G. 1987a. Silver ions trigger Ca2+ release by interaction with the (Ca2+−Mg2+)-ATPase in reconstituted systems.J. Biol. Chem. 262:7676–7679PubMedGoogle Scholar
  21. Guillain, F., Gingold, M.P., Buschlen, S., Champeil, P. 1980. A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase.J. Biol. Chem. 255:2072–2076PubMedGoogle Scholar
  22. Hasselbach, W., Makinose, M. 1961. Die calcium pumpe der erschlaffungsgrana des muskels und ihre abhangigkeit von der ATP-spaltung.Biochem. Z. 333:518–528PubMedGoogle Scholar
  23. Hille, B. 1982. Ionic Channels of Excitable Membranes. Sinauer Associates Inc., Sunderland, MassachusettsGoogle Scholar
  24. Hymel, L., Maurer, A., Berenski, C., Jung, C.Y., Fleischer, S. 1984. Target size of calcium pump protein from skeletal muscle sarcoplasmic reticulum.J. Biol. Chem. 259:4890–4895PubMedGoogle Scholar
  25. Ikemoto, N. 1982. Structure and function of the calcium pump protein of sarcoplasmic reticulum.Annu. Rev. Physiol. 44:297–317PubMedGoogle Scholar
  26. Ikemoto, N., Miyao, A., Kurobe, Y. 1981. Further evidence for an oligomeric calcium pump by sarcoplasmic reticulum.J. Biol. Chem. 256:10809–10814PubMedGoogle Scholar
  27. Inesi, G., Scales, D. 1974. Tryptic cleavage of sarcoplasmic reticulum protein.Biochemistry 13:3298–3306PubMedGoogle Scholar
  28. Inui, M., Saito, A., Fleischer, S. 1987. Purification of the ryanodine receptor and identity with feet structures of junctional terminal cisternae of sarcoplasmic reticulum from fast skeletal muscle.J. Biol. Chem. 262:1740–1747PubMedGoogle Scholar
  29. Klip, A., Reithmeier, R.A.F., MacLennan, D.H. 1980. Resolution of enzymes of biological transport. 13. Alignment of the major tryptic fragments of the adenosine triphosphatase from sarcoplasmic reticulum.J. Biol. Chem. 255:6562–6568PubMedGoogle Scholar
  30. Krasne, S., Szabo, G., Eisenman, G. 1971. Freezing and melting of lipid bilayers and the mode of action of nonactin, valinomycin and gramicidin.Science 174:412–415PubMedGoogle Scholar
  31. Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685Google Scholar
  32. Lattanzio, F.A., Schlatter, R.G., Nicar, M., Campbell, K.P., Sutko, J.L. 1987. The effects of ryanodine on passive calcium fluxes across sarcoplasmic reticulum membranes.J. Biol. Chem. 262:2711–2718PubMedGoogle Scholar
  33. McFarland, B., Inesi, G. 1971. Solubilization of sarcoplasmic reticulum with Triton X-100.Arch. Biochem. Biophys. 145:456–464PubMedGoogle Scholar
  34. MacLennan, D.H. 1970. Purification and properties of an adenosine triphosphatase from sarcoplasmic reticulum.J. Biol. Chem. 245:4508–4518PubMedGoogle Scholar
  35. McLennan, D.H., Brandl, C.J., Korczak, B., Green, N.M. 1985. Amino-acid sequence of a Ca2++Mg2+-dependent ATPase from rabbit muscle sarcoplasmic reticulum, deduced from its complementary DNA sequence.Nature (London) 316:696–700Google Scholar
  36. MacLennan, D.H., Holland, P.C. 1975. Calcium transport in sarcoplasmic reticulum.Annu. Rev. Biophys. Bioeng. 4:377–404PubMedGoogle Scholar
  37. McWhirter, J.M., Gould, G.W., East, J.M., Lee, A.G. 1987. A kinetic model for Ca2+ efflux mediated by the Ca2++Mg2+-activated ATPase of sarcoplasmic reticulum.Biochem. J. 245:713–722PubMedGoogle Scholar
  38. Marsh, B.B. 1952. The effects of adenosine triphosphate on the fibre volume of a muscle homogenate.Biochim. Biophys. Acta 9:247–260PubMedGoogle Scholar
  39. Martin, D.W., Tanford, C., Reynolds, J.A. 1984. Monomeric solubilized sarcoplasmic reticulum calcium pump protein. Demonstration of Ca2+ binding and dissociation coupled to ATP hydrolysis.Proc. Natl. Acad. Sci. USA 81:6623–6626PubMedGoogle Scholar
  40. Martonosi, A., Donley, J., Halpin, R.A. 1968. Sarcoplasmic reticulum. The role of phospholipids in the adenosine triphosphatase activity and Ca2+ transport.J. Biol. Chem. 243:61–70PubMedGoogle Scholar
  41. Martonosi, A., Feretos, R. 1964. Sarcoplasmic reticulum. The uptake of Ca2+ by sarcoplasmic reticulum fragments.J. Biol. Chem. 239:648–657PubMedGoogle Scholar
  42. Martonosi, A., Halpin, R. 1971. Sarcoplasmic reticulum. X. The protein composition of sarcoplasmic reticulum membrane.Arch. Biochem. Biophys. 144:66–77PubMedGoogle Scholar
  43. Martonosi, M.A. 1974. Thermal analysis of sarcoplasmic reticulum membranes.FEBS Lett. 47:327–329PubMedGoogle Scholar
  44. Mészáros, L.G., Ikemoto, N. 1985. Ruthenium red and caffeine affect the Ca2+-ATPase of the sarcoplasmic reticulum.Biochem. Biophys. Res. Commun. 127:836–842PubMedGoogle Scholar
  45. Migala, A., Agostini, B., Hasselbach, W. 1973. Tryptic fragmentation of the calcium transport system in the sarcoplasmic reticulum.Z. Naturforsch. 28:178–182Google Scholar
  46. Millman, M.S. 1980. A thermal transition of passive calcium efflux in fragmented sarcoplasmic reticulum.Membr. Biochem. 3:271–290PubMedGoogle Scholar
  47. Mrak, R.E., Fleischer, S. 1982. Normal function in sarcoplasmic reticulum from the mice with muscular dystrophy.Muscle and Nerve 5:143–151PubMedGoogle Scholar
  48. Mueller, P., Rudin, D.O. 1968. Action potentials induced in bimolecular lipid membranes.Nature (London) 217:713–719Google Scholar
  49. Murphy, A.J. 1976. Sulfhydryl group modification of sarcoplasmic reticulum membranes.Biochemistry 15:4492–4496PubMedGoogle Scholar
  50. Murphy, A.J. 1981. Kinetics of inactivation of the ATPase of sarcoplasmic reticulum by dicyclohexylcarbodiimide.J. Biol. Chem. 256:12046–12050PubMedGoogle Scholar
  51. Pascolini, D., Asturias, F., Blasie, J.K. 1987. Moderate resolution profile structure of the sarcoplasmic reticulum membrane under “low” temperature conditions.Biophys. J. 51:347aGoogle Scholar
  52. Pick, U., Karlish, J.D. 1982. Regulation of the conformational transition in the Ca-ATPase from sarcoplasmic reticulum by pH, temperature and calcium ions.J. Biol. Chem. 257:6120–6126PubMedGoogle Scholar
  53. Pick, U., Racker, E. 1979. Inhibition of the (Ca2+) ATPase from sarcoplasmic reticulum by dicyclohexylcarbodiimide: Evidence for location of the Ca2+ binding site in a hydrophobic region.Biochemistry 18:108–113PubMedGoogle Scholar
  54. Scales, D., Inesi, G. 1976. Assembly of ATPase protein in sarcoplasmic reticulum membranes.Biophys. J. 16:735–751PubMedGoogle Scholar
  55. Scott, T.L., Shamoo, A.E. 1982. Disruption of energy transduction in sarcoplasmic reticulum by trypsin cleavage of (Ca2++Mg2+)-ATPase.J. Membrane Biol. 64:137–144Google Scholar
  56. Scott, T.L., Shamoo, A.E. 1984. Distinction of the roles of the two high affinity calcium sites in the functional activities of the Ca2+-ATPase of sarcoplasmic reticulum.Eur. J. Biochem. 143:427–436PubMedGoogle Scholar
  57. Shamoo, A.E., Herrmann, T.R., Gangola, R., Joshi, N.B. 1987. Biophysical aspects of Ca2+-transport sites in skeletal and cardiac sarcoplasmic reticulum (Ca2++Mg2+)-ATPase.In: Heart Function and Metabolism. N.S. Dhalla, G.N. Pierce, and R.E. Beamish, editors, pp. 221–241. Martinus NijhoffGoogle Scholar
  58. Shamoo, A.E., Ryan, T.E. 1975. Isolation of ionophores from ion-transport systems.Ann. N.Y. Acad. Sci. 264:83–97PubMedGoogle Scholar
  59. Shamoo, A.E., Ryan, T.E., Stewart, P.S., MacLennan, D.H. 1976. Localization of ionophore activity in a 20,000-dalton fragment of the adenosine triphosphatase of sarcoplasmic reticulum.J. Biol. Chem. 251:4147–4154PubMedGoogle Scholar
  60. Shamoo, A.E., Scott, T.L., Ryan, T.E. 1977. Active calcium transport via coupling between the enzymatic and the ionophoric sites of Ca2++Mg2+-ATPase.J. Supramol. Struct. 6:345–353PubMedGoogle Scholar
  61. Silva, J.L., Verjovski-Almeida, S. 1985. Monomer-dimer association constant of solubilized sarcoplasmic reticulum ATPase.J. Biol. Chem. 260:4764–4769PubMedGoogle Scholar
  62. Stewart, P.S., MacLennan, D.H. 1974. Surface particles of sarcoplasmic reticulum membrane. Structural features of the adenosine triphosphatase.J. Biol. Chem. 249:985–993PubMedGoogle Scholar
  63. Stewart, P.S., MacLennan, D.H., Shamoo, A.E. 1976. Isolation and characterization of tryptic fragments of the adenosine triphosphatase of sarcoplasmic reticulum.J. Biol. Chem. 251:712–719PubMedGoogle Scholar
  64. Tanford, C. 1984. Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump.Crit. Rev. Biochem. 17:123–151Google Scholar
  65. Taylor, K., Dux, L., Martonosi, A. 1984. Structure of the vanadate-induced crystals of sarcoplasmic reticulum Ca-ATPase.J. Mol. Biol. 174:193–204PubMedGoogle Scholar
  66. Thorley-Lawson, D.A., Green, N.M. 1973. Studies on the location and orientation of proteins in the sarcoplasmic reticulum.Eur. J. Biochem. 40:403–413PubMedGoogle Scholar
  67. Toogood, K.C., Folsom, B., Topping, T., McCutchan, H., Dolejsi, M. J., Johns, S., Stuart, G., Dunker, A.K. 1983. Evidence that trypsin digestion exposes a channel in the sarcoplasmic reticulum membrane.Membr. Biochem. 5:49–75PubMedGoogle Scholar
  68. Tosteson, D.C., Andreoli, T.E., Tieffenberg, M., Cook, P.J. 1968. The effects of macrocyclic compounds on cation transport in sheep red cells and thin and thick lipid membranes.J. Gen. Physiol. 51:373s-384sPubMedGoogle Scholar
  69. Weber, A., Herz, R., Reiss, I. 1966. Study of the kinetics of calcium transport by isolated fragmented sarcoplasmic reticulum.Biochem. Z. 345:329–369Google Scholar
  70. Weber, K., Osborn, M. 1969. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis.J. Biol. Chem. 244:4406–4612PubMedGoogle Scholar
  71. Yamada, S., Ikemoto, N. 1978. Distinction of thiols involved in the specific reaction steps of the Ca2+-ATPase of the sarcoplasmic reticulum.J. Biol. Chem. 253:6801–6807PubMedGoogle Scholar
  72. Yamanaka, N., Deamer, D.W. 1976. Protease digestion of membranes. Ultrastructural and biochemical effects.Biochim. Biophys. Acta 426:132–147PubMedGoogle Scholar
  73. Yount, R.G., Babcock, D., Ballentyne, W., Ojala, D. 1971. Adenylyl imidodiphosphate, an adenosine triphosphate analogue containing a P−N−P linkage.Biochemistry 10:2484–2489PubMedGoogle Scholar

Copyright information

© Springer-Verlag New York Inc. 1987

Authors and Affiliations

  • Jun-Ling Huang
    • 1
  • Traci B. Topping
    • 1
  • Zhaoping He
    • 1
  • Brian Folsom
    • 1
  • A. Keith Dunker
    • 1
  1. 1.Biochemistry/Biophysics Program and Department of ChemistryWashington State UniversityPullman

Personalised recommendations