Sites of labeling with N-(3-pyrene)maleimide on Ca(2+)-transporting ATPase of the sarcoplasmic reticulum.
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 Ca(2+)-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 Ca(2+)-transport caused by PMI.
Suzuki
T
T
Department of Physiological Chemistry, Tokyo Metropolitan Institute of Medical Science.
Kawakita
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003593:Cytoplasm / Q000201:enzymology; D005456:Fluorescent Dyes / Q000378:metabolism* / Q000494:pharmacology; D007425:Intracellular Membranes / Q000201:enzymology; D008301:Maleimides / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013439:Sulfhydryl Reagents / Q000378:metabolism / Q000494:pharmacology
D005456:Fluorescent Dyes; D008301:Maleimides; D013439:Sulfhydryl Reagents; C003944:N-(3-pyrene)maleimide; D000252:Calcium-Transporting ATPases