Sites of Labeling with N-(3-Pyrene) Maleimide on Ca²⁺-Transporting ATPase of the Sarcoplasmic Reticulum

抄録

The characteristics of the interaction between N-(3-pyrene)maleimide (PMI) and the sarcoplasmic reticulum (SR) membranes were investigated, and the sites of labeling with PMI on Ca²⁺-transporting ATPase were identified. PMI was dissolved in the membrane lipids before reacting with the ATPase protein. The measurement of resonance energy transfer from PMI to 1-(dimethylaminophenyl) -6-phenyl-1, 3, 5-hexatriene revealed that the pyrene moiety of PMI stayed in the lipid layer after it had been covalently attached to the ATPase molecule. PMI-labeled SR membranes at an average labeling density of 1 mol PMI/mol ATPase were digested with trypsin, and the labeled peptides were purified through a series of reversed-phase HPLC procedures on C₁₈ and C₄ columns. The amino acid analysis of the purified peptides revealed multiple cysteine residues mainly distributed over the C-terminal half of the cytoplasmic domain of the ATPase molecule as the targets of PMI. This implied that PMI molecules mediated cross-linking between the cytoplasmic domain of the ATPase molecule and the membranes. The distortion of the structure of the former due to this cross-linking may explain the uncoupling of ATP-splitting from Ca²⁺-transport caused by PMI.