A monoclonal antibody recognizes an epitope in the first extracellular loop of the plasma membrane Ca2+ pump.
A monoclonal antibody against the human erythrocyte Ca2+ pump (1E4) reacted with the enzyme in intact erythrocytes. Using trypsinized preparations of the pump the antibody only reacted with the N-terminal fragments of 33.5 and 35 kDa. The fragments span from the N terminus (35 kDa) or from residue 19 (33.5 kDa) to residue 314 of the hPMCA4 isoform of the pump. Exhaustive degradation with a number of agents produced smaller peptides which reacted with the antibody. Sequencing analysis on two chymotryptic fragments of 8.8 and 13.5 kDa identified the epitope in an approximately 80-residue domain beginning with Gly-81. Two peptides corresponding to the putative extramembrane portions of this region of the pump were synthesized. The antibody reacted with one of them, spanning residues Phe-121 to Gly-152 and containing the first putative external loop of the pump. Peptides corresponding to overlapping portions of this peptide were synthesized, leading to the location of the epitope in a 13-residue sequence (Glu-130 to Glu-142) in the first predicted extracellular loop (Verma, A. K., Filoteo, A. G., Stanford, D. R., Wieben, E. D., Strehler, E. E., Fischer, R., Heim, R., Vogel, G., Mathews, S., Strehler-Page, M-A., James, P., Vorherr, T., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 14152-14159).
Feschenko
M S
MS
Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.
Zvaritch
E I
EI
Hofmann
F
F
Shakhparonov
M I
MI
Modyanov
N N
NN
Vorherr
T
T
Carafoli
E
E
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015153:Blotting, Western; D000252:Calcium-Transporting ATPases / Q000276:immunology* / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D004910:Erythrocyte Membrane / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D014357:Trypsin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000939:Epitopes; D002918:Chymotrypsin; D014357:Trypsin; D000252:Calcium-Transporting ATPases