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

Tryptic peptides of Ca-ATPase in E1 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 preferential hydrophobic labeling in E2 forms was found to be localized in the A1 tryptic fragment.