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.