title: The plasma membrane Ca2+ pump contains a site that interacts with its calmodulin-binding domain.
abstract: A synthetic, 28-residue peptide derived from the calmodulin-binding sequence of the plasma membrane Ca2+ pump (C28W) inhibits the ATPase activity of a calpain-produced, truncated fragment of the enzyme. The fragment, which has lost the calmodulin-binding domain, has a molecular mass of 124 kDa and is fully active in the absence of calmodulin. Replacement of Trp-8 in the peptide by an Ala decreases the overall inhibitory activity, while replacement with a Tyr increases it. However, at very low peptide concentrations the effect of Tyr replacement disappears. The synthetic peptide has been made photoactivatable by replacing Phe in position 9 with a synthetic phenylalanine analogue containing a diazirine group and was radioactively labeled by coupling a [3H]acetyl function to its N terminus. After cross-linking with the derivatized peptide, the 124-kDa fragment has been proteolyzed with either Lys-C, Asp-N, or V8 proteases, and the fragment(s) have been separated. Partial sequencing of the cross-linked, radioactive peptides has identified a site of the pump located C terminally to the phosphoenzyme-forming aspartic acid, spanning residues 537-544 of the hPMCA4 isoform of the enzyme. It is concluded that this sequence is part of a site which binds the calmodulin-binding domain of the pump.
authors.affiliation: Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.
authors.forename: R
authors.initials: R
authors.lastname: Falchetto
authors.forename: T
authors.initials: T
authors.lastname: Vorherr
authors.forename: J
authors.initials: J
authors.lastname: Brunner
authors.forename: E
authors.initials: E
authors.lastname: Carafoli
mesh_terms: D000595:Amino Acid Sequence; D001692:Biological Transport; D015220:Calcium Channels / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D004910:Erythrocyte Membrane / Q000378:metabolism; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D008969:Molecular Sequence Data; D016415:Sequence Alignment
chemical_list: D015220:Calcium Channels; D002147:Calmodulin; D000252:Calcium-Transporting ATPases