Summary
Conformational states in sarcoplasmic reticulum Ca2+-ATPase have been examined by tryptic and chymotryptic cleavage. High affinity Ca2+ binding (E1 state) exposes a peptide bond in the A fragment of the polypeptide chain to trypsin. Absence of Ca2+ (E2 state) exposes bonds in the B fragment, which are protected by binding of Mg2+ or ATP. After phosphorylation from ATP the tryptic cleavage pattern depends on the predominant phosphoenzyme species present. ADP-sensitive E1P and ADP-insensitive E2P have cleavage patterns identical to those of unphosphorylated E1 and E2, respectively, indicating that two major conformational states are involved in Ca2+ translocation. The transition from E1P to E2P is inhibited by secondary tryptic splits in the A fragment, suggesting that parts of this fragment are of particular importance for the energy transduction process.
The tryptic cleavage patterns of phosphorylated forms of detergent solubilized monomeric Ca2+-ATPase were similar to those of the membrane-bound enzyme, indicating that Ca2+ translocation depends mainly on structural changes within a single peptide chain. On the other hand, the protection of the second cleavage site as observed after vanadate binding to membranous Ca2+-ATPase could not be achieved in the soluble monomeric enzyme. Shielding of this peptide bond may therefore be due to protein-protein interactions in the semicrystalline state of the vanadate-bound Ca2+-ATPase in membranous form.
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References
Allen, G., Trinnaman, B.J., Green, N.M. 1980. The primary structure of the calcium ion-transporting adenosine triphosphatase protein of rabbit skeletal sarcoplasmic reticulum.Biochem. J. 187:591–616
Andersen, J.P., Fellmann, P., Møller, J.V., Devaux, P.F. 1981. Immobilization of a spin-labeled fatty acid chain covalently attached to Ca2+-ATPase from sarcoplasmic reticulum suggests an oligomeric structure.Biochemistry 20:4928–4936
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. 260:371–380
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. 257:8300–8307
Andersen, J.P., Skriver, E., Mahrous, T.S., Møller, J.V. 1983. Reconstitution of sarcoplasmic reticulum Ca2+-ATPase with excess lipid: Dispersion of the pump units.Biochim. Biophys. Acta 728:1–10
Avruch, J., Fairbanks, G. 1972. Demonstration of a phosphopeptide intermediate in the Mg2+-dependent, Na+- and K+-stimulated adenosine triphosphatase reaction of the erythrocyte membrane.Proc. Natl. Acad. Sci. USA 69:1216–1220
Champeil, P., Bastide, F., Taupin, C., Gary-Bobo, C.M. 1976. Spin labelled sarcoplasmic reticulum vesicles: Ca2+-induced spectral changes.FEBS Lett. 63:270–272
Champeil, P., Gingold, M.P., Guillain, F., Inesi, G. 1983. Effect of magnesium on the calcium-dependent transient kinetics of sarcoplasmic reticulum ATPase studied by stopped flow fluorescence and phosphorylation.J. Biol. Chem. 258:4453–4458
Coan, C., Verjovski-Almeida, S., Inesi, G. 1979. Ca2+ regulation of conformational states in the transport cycle of spin-labelled sarcoplasmic reticulum ATPase.J. Biol. Chem. 254:2968–2974
De Meis, L. 1981. The Sarcoplasmic Reticulum. John Wiley & Sons, New York
Dupont, Y. 1976. Fluorescence studies of the sarcoplasmic reticulum calcium pump.Biochem. Biophys. Res. Commun. 71:544–550
Dupont, Y. 1980. Occlusion of divalent cations in the phosphorylated calcium pump of sarcoplasmic reticulum.Eur. J. Biochem. 109:231–238
Dupont, Y., Bennett, N., Lacapere, J. 1982. ATP-induced conformational transitions of the Ca2+-ATPase of sarcoplasmic reticulum.Ann. N.Y. Acad. Sci. 402:569–572
Dupont, Y., Pougeois, R. 1983. Evaluation of H2O activity in the free or phosphorylated catalytic site of Ca2+-ATPase.FEBS Lett. 156:93–98
Dux, L., Martonosi, A. 1983. Ca2+-ATPase crystals in sarcoplasmic reticulum: The effect of trypsin digestion.J. Biol. Chem. 258:10111–10115
Green, N.M., Allen, G., Hebdon, G.M. 1980. Structural relationship between the calcium- and magnesium-transporting ATPase of sarcoplasmic reticulum and the membrane.Ann. N.Y. Acad. Sci. 358:149–158
Guillain, F., Champeil, P., Lacapere, J., Gingold, M.P. 1981. Stopped flow and rapid quenching measurement of the transient steps induced by calcium binding to sarcoplasmic reticulum adenosine triphosphatase.J. Biol. Chem. 256:6140–6147
Guillain, F., Gingold, M.P., Büschlen, S., Champeil, P. 1980. A direct fluorescence study of the transient steps induced by calcium binding to sarcoplasmic reticulum ATPase.J. Biol. Chem. 255:2072–2076
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. 259:4890–4895
Ikemoto, N., Sreter, F.A., Gergely, J. 1971. Structural features of the vesicles of FSR: Lack of functional role in Ca2+-uptake and ATPase activity.Arch. Biochem. Biophys. 147:571–582
Imamura, Y., Saito, K., Kawakita, M. 1984. Conformational change of Ca2+, Mg2+-adenosine triphosphatase of sarcoplasmic reticulum upon binding of Ca2+ and adenyl-5′-ylimidodiphosphate as detected by trypsin sensitivity analysis.J. Biochem. 95:1305–1313
Inesi, G., Scales, D. 1974. Tryptic cleavage of sarcoplasmic reticulum protein.Biochemistry 13:3298–3306
Jørgensen, P.L. 1983. Mechanism of the Na+, K+ Pump. Protein structure and conformations of the pure (Na++K+)-ATPase.Biochim. Biophys. Acta 694:27–68
Jørgensen, P.L., Petersen, J. 1975. Purification and characterization of (Na+, K+)-ATPase: V. Conformational changes in the enzyme. Transitions between the Na-form and the K-form studied with tryptic digestion as a tool.Biochim. Biophys. Acta 401:399–415
Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature (London) 227:680–685
Lüdi, J., Hasselbach, W. 1984. Separation of the tryptic fragments of sarcoplasmic reticulum ATPase with high performance liquid chromatography. Identification of the calcium binding site.FEBS Lett. 167:33–36
MacLennan, D.H., Reithmeier, A.F. 1982. The structure of the Ca2+/Mg2+-ATPase of sarcoplasmic reticulum.In: Membranes and Transport. A.N. Martonosi, editor. pp. 567–571. Plenum, New York
Marcsek, Z., Nelson, R., Ikemoto, N. 1983. Bivalent cation dependent conformational changes of the sarcoplasmic reticulum Ca2+-ATPase modify the second tryptic cleavage site. Abstracts of the 15th FEBS Meeting, Brussels 1983
Martin, D.W., Tanford, C., Reynolds, J.A. 1984. Monomeric solubilized sarcoplasmic reticulum Ca pump protein: Demonstration of Ca binding and dissociation coupled to ATP hydrolysis.Proc. Natl. Acad. Sci. USA 81:6623–6626
Martonosi, A.N., Beeler, T.J. 1983. Mechanism of Ca2+-transport by sarcoplasmic reticulum.In: Handbook of Physiology. Section 10: Skeletal Muscle. pp. 417–485. American Physiological Society, Bethesda
Migala, A., Agostini, B., Hasselbach, W. 1973. Tryptic fragmentation of the calcium transport system in the sarcoplasmic reticulum.Z. Naturforsch. 28c:178–182
Møller, J.V., Andersen, J.P., Maire, M. le 1982. The sarcoplasmic reticulum Ca2+-ATPase.Mol. Cell. Biochem. 42:83–107
Møller, J.V., Lind, K.E., Andersen, J.P. 1980. Enzyme kinetics and substrate stabilization of detergent-solubilized and membraneous (Ca2++Mg2+)-activated ATPase from sarcoplasmic reticulum.J. Biol. Chem. 255:1912–1920
Murphy, A.J. 1976.Sulfhydryl group modification of sarcoplasmic reticulum membranes.Biochemistry 15:4492–4496
Pick, U., Karlish, S.J.D. 1982. Regulation of the conformational transition in the Ca-ATPase from sarcoplasmic reticulum by pH, temperature and calcium ions.J. Biol. Chem. 257:6120–6126
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 18:108–113
Pickart, C.M., Jencks, W.P. 1984. Energetics of the calcium-transporting ATPase.J. Biol. Chem. 259:1629–1643
Poduslo, J.F., Rodbard, D. 1980. Molecular weight estimation using sodium dodecyl sulfate-pore gradient electrophoresis.Anal. Biochem. 101:394–406
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. 143:427–436
Stewart, P.S., MacLennan, D.H. 1974. Surface particles of sarcoplasmic reticulum membranes: Structural features of the adenosine triphosphatase,J. Biol. Chem. 249:985–993
Takisawa, H., Makinose, M. 1983. Occlusion of calcium in the ADP-sensitive phosphoenzyme of the adenosine triphosphatase of sarcoplasmic reticulum,J. Biol. Chem. 258:2986–2992
Tanford, C. 1984. Twenty questions concerning the reaction cycle of the sarcoplasmic reticulum calcium pump.CRC Crit. Rev. Biochem. 17:123–151
Taylor, K., Dux, L., Martonosi, A. 1984. Structure of the vanadate-induced crystals of sarcoplasmic reticulum Ca2+-ATPase.J. Mol. Biol. 174:193–204
Thorley-Lawson, D.A., Green, N.M. 1973. Studies on the location and orientation of proteins in the sarcoplasmic reticulum.Eur. J. Biochem. 40:403–413
Vianna, A.L. 1975. Interaction of calcium and magnesium in activating and inhibiting the nucleoside triphosphatase of sarcoplasmic reticulum vesicles.Biochim. Biophys. Acta 410:389–406
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. 253:6801–6807
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Andersen, J.P., Jørgensen, P.L. Conformational states of sarcoplasmic reticulum Ca2+-ATPase as studied by proteolytic cleavage. J. Membrain Biol. 88, 187–198 (1985). https://doi.org/10.1007/BF01868432
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DOI: https://doi.org/10.1007/BF01868432