The calmodulin binding domain of the plasma membrane Ca2+ pump interacts both with calmodulin and with another part of the pump.
Synthetic peptides corresponding to the calmodulin-binding domain of the human erythrocyte Ca2+ pump were prepared representing residues 2-29 (C28W), 2-21 (C20W), 2-16 (C15W), and 16-29 (C14) of the sequence (James, P., Maeda, M., Fisher, R., Verma, A. K., Krebs, J., Penniston, J. T., and Carafoli, E. (1988) J. Biol. Chem. 263, 2905-2910). Peptides C28W, C20W, and C15W bound to calmodulin with an apparent 1:1 stoichiometry in the presence of Ca2+ and inhibited the activation of the Ca2+ pump by calmodulin, while C14 was ineffective. Substituting tyrosine (C28Y) or alanine (C28A) for the tryptophan residue lowered the affinity for calmodulin. The estimated Kd values for the calmodulin-peptide complexes were 0.1 nM for C28W, 5-15 nM for C20W, C28Y, and C28A, and 700-1700 nM for C15W. The Ca2+ pump in inside-out erythrocyte membrane vesicles was activated by proteolytic removal of the endogenous calmodulin-binding domain. Addition of C20W or C28W then inhibited calmodulin-independent Ca2+ transport, while a calmodulin-binding peptide from another enzyme had no effect. The inhibition of the pump by C20W was purely competitive with Ca2+, while C28W decreased the Vmax and increased the K1/2 for Ca2+, restoring the pump activity nearly to its low basal level. The results suggest that a calmodulin-binding peptide from any enzyme has two kinds of specificity: it shares with peptides from other enzymes the ability to bind to calmodulin, but only it has the specificity to interact with its own (proteolytically activated) enzyme.
Enyedi
A
A
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
Vorherr
T
T
James
P
P
McCormick
D J
DJ
Filoteo
A G
AG
Carafoli
E
E
Penniston
J T
JT
D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000097:blood; D000252:Calcium-Transporting ATPases / Q000097:blood* / Q000302:isolation & purification; D002147:Calmodulin / Q000378:metabolism*; D004910:Erythrocyte Membrane / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding
D002147:Calmodulin; D010455:Peptides; D000252:Calcium-Transporting ATPases; D002118:Calcium