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

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Abstract
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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