The epitope for monoclonal antibody A20 (amino acids 870-890) is located on the luminal surface of the Ca2(+)-ATPase of sarcoplasmic reticulum.
The epitope for monoclonal antibody A20 was mapped to amino acids 870-890 of the Ca2(+)-ATPase of rabbit fast-twitch skeletal muscle sarcoplasmic reticulum. The antibody did not react with the epitope in intact sarcoplasmic reticulum vesicles but reacted with the epitope when the vesicles were solubilized with the detergent C12E8 or made permeable by incubation in a hypotonic medium. By contrast, antibody A52, which binds to a cytoplasmic epitope consisting of amino acids 657-672, reacted with the Ca2(+)-ATPase in vesicular, permeabilized vesicular, and C12E8-solubilized states. These results clearly demonstrate that antibody A20 binds to a luminal epitope and provide the first demonstration that a specific segment of the Ca2(+)-ATPase is located on the luminal surface of the sarcoplasmic reticulum. These results are consistent with, and support, our model for folding of the Ca2(+)-ATPase (Brandl, C. J., Korczak, B., Green, N. M., and MacLennan, D. H. (1986) Cell 44, 597-607) in which residues 657-672 were proposed to form part of the cytoplasmic nucleotide binding domain, while residues 870-890 were proposed to form a luminal loop between proposed transmembrane sequences M7 and M8.
Banting and Best Department of Medical Research, Charles H. Best Institute, University of Toronto, Ontario, Canada.
D M
DM
Clarke
T W
TW
Loo
D H
DH
MacLennan
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex / Q000032:analysis; D000252:Calcium-Transporting ATPases / Q000276:immunology* / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D007700:Kinetics; D009132:Muscles / Q000201:enzymology*; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D000939:Epitopes; D000252:Calcium-Transporting ATPases