Structure-function studies on calcium transporting proteins within the adductor muscle of the sea scallop

An unusual pattern of Ca2+-dependent tryptic cleavage sites in the cytoplasmic domains of scallop Ca2+-ATPase allowed it to be shown that binding of the phosphate moieties of nucleotides and nucleotide analogues or orthophosphate to the catalytic site of the E 1 enzyme induced a previously undescribed intermediate in which the enzyme is repositioned such that the terminal phosphoryl group of ATP is prepared for transfer to the catalytic site. Covalent phosphorylation of the Ca 2+-ATPase to form E2-P led to increased stabilization of the enzyme against proteolysis by trypsin. Kinetic data from the scallop Ca 2+-ATPase were consistent with the presence of both a low and high affinity ATP binding site.; The cytosolic f loop of the scallop Na+-Ca2+-Exchanger, together with much of the N-terminal transmembrane region, was sequenced by RT-PCR. The scallop exchanger f loop possessed three Protein Kinase A (PK-A) consensus sequences. One of these (K600RGSV) was a plausible candidate for a site phosphorylated by PK-A detected in previous studies on the native scallop exchanger. Regions of the scallop exchanger f loop that correspond closely to previously described 37k Da, 16k Da, and 19k Da tryptic peptides were expressed as fusion proteins. In the 37k Da fusion proteins, there was a significant (∼25%) increase in secondary structure content when Ca2+ were bound, indicating that functional Ca 2+-binding sites were present. The 37k Da fusion protein and the isolated 37k Da peptide could be phosphorylated by PK-A in the presence of EGTA. Site-directed mutagenesis identified Ser603 as the residue in the exchanger portion of the fusion protein that was phosphorylated by PK-A. When PK-A treatment of the isolated 37k Da peptide was carried out in the presence of Ca 2+, no phosphorylation was observed.