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

Abstract

Summary Tryptic peptides of Ca-ATPase in E_t 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.