Binding of calcium by calmodulin: influence of the calmodulin binding domain of the plasma membrane calcium pump.
The interaction between calmodulin and synthetic peptides corresponding to the calmodulin binding domain of the plasma membrane Ca2+ pump has been studied by measuring Ca2+ binding to calmodulin. The largest peptide (C28W) corresponding to the complete 28 amino acid calmodulin binding domain enhanced the Ca2+ affinity of calmodulin by more than 100 times, implying that the binding of Ca2+ increased the affinity of calmodulin for the peptide by more than 10(8) times. Deletion of the 8 C-terminal residues from peptide C28W did not decrease the affinity of Ca2+ for the high-affinity sites of calmodulin, but it decreased that for the low-affinity sites. A larger deletion (13 residues) decreased the affinity of Ca2+ for the high-affinity sites as well. The data suggest that the middle portion of peptide C28W interacts with the C-terminal half of calmodulin. Addition of the peptides to a mixture of tryptic fragments corresponding to the N- and C-terminal halves of calmodulin produced a biphasic Ca2+ binding curve, and the effect of peptides was different from that on calmodulin. The result shows that one molecule of peptide C28W binds both calmodulin fragments. Interaction of the two domains of calmodulin through the central helix is necessary for the high-affinity binding of four Ca2+ molecules.
Yazawa
M
M
Department of Chemistry, Faculty of Science, Hokkaido University, Sapporo, Japan.
Vorherr
T
T
James
P
P
Carafoli
E
E
Yagi
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry*; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D008974:Mollusca; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014357:Trypsin
D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D010455:Peptides; D014357:Trypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium