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.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D014357:Trypsin / Q000378:metabolism*
D000596:Amino Acids; D010446:Peptide Fragments; D014357:Trypsin; D000252:Calcium-Transporting ATPases