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Localization of E1–E2 conformational transitions of sarcoplasmic reticulum Ca-ATPase by tryptic cleavage and hydrophobic labeling

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Summary

Tryptic peptides of Ca-ATPase in Et and E2 conformational states (Andersen, J. P., Jørgensen, P. L.,J. Membrane Biol. 88:187–198 (1985)) have been isolated by size exclusion high performance liquid chromatography in sodium dodecyl sulfate. This permitted unambiguous localization of a conformational sensitive tryptic split at Arg 198 by N-terminal amino acid sequence analysis. Other splits at Arg 505 and at Arg 819-Lys 825 were insensitive to E1–E2 transitions. Tryptic cleavage of Ca-ATPase after phosphorylation by inorganic phosphate showed that this enzyme form has a conformation similar to that of the vanadate-bound E2 state, both in membranous and in soluble monomeric Ca-ATPase.

Hydrophobic labeling of Ca-ATPase in sarcoplasmic reticulum vesicles with the photoactivable reagent trifluoromethyl-[125I]iodophenyl-diazirine indicated that E2 and E2V states are more exposed to the membrane phase than E1 and E1P (Ca2+-occluded) states. The preferetial hydrophobic labeling in E2 forms was found to be localized in the A1 tryptic fragment.

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Authors and Affiliations

  1. Institute of Physiology, Aarhus University, DK-8000, Aarhus C, Denmark

    Jens P. Andersen, Bente Vilsen & Peter L. Jørgensen

  2. Department of Biology, Clarkson University, 13676, Potsdam, New York

    John H. Collins

Authors
  1. Jens P. Andersen
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  2. Bente Vilsen
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  3. John H. Collins
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  4. Peter L. Jørgensen
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Andersen, J.P., Vilsen, B., Collins, J.H. et al. Localization of E1–E2 conformational transitions of sarcoplasmic reticulum Ca-ATPase by tryptic cleavage and hydrophobic labeling. J. Membrain Biol. 93, 85–92 (1986). https://doi.org/10.1007/BF01871021

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