Identification of two domains which mediate the binding of activating phospholipids to the plasma-membrane Ca2+ pump.
The stimulation of the purified human erythrocyte calcium pump by acidic phospholipids was investigated using synthetic peptides corresponding to a putative phospholipid-responsive domain [Zvaritch, E., James, P., Vorherr, T., Falchetto, R., Modyanov, N. & Carafoli, E. (1990) Biochemistry 29, 8070-8076] and to the calmodulin-binding domain of the pump. The peptides interfered with the activation of the enzyme by phosphatidylserine and phosphatidic acid in competition assays. The peptide corresponding to the calmodulin-binding domain was found to be the most efficient antagonist. Direct binding measurements using fluorescent derivatives of the peptides confirmed the interaction between the acidic phospholipids and the peptides, and fluorescence titrations of dansylated calmodulin with the purified ATPase showed a direct effect of acidic phospholipids on calmodulin binding. A proteolyzed preparation of the Ca(2+)-ATPase lacking the calmodulin-binding domain confirmed that the phospholipid-induced stimulation is mediated by two sites, one located in the C-terminal portion of the previously identified 44-amino-acid phospholipid-responsive domain, the other in the calmodulin-binding domain.
Brodin
P
P
Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich.
Falchetto
R
R
Vorherr
T
T
Carafoli
E
E
D000595:Amino Acid Sequence; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003619:Dansyl Compounds / Q000378:metabolism; D004910:Erythrocyte Membrane / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism*; D013050:Spectrometry, Fluorescence
D002147:Calmodulin; D003619:Dansyl Compounds; D010455:Peptides; D010743:Phospholipids; D000252:Calcium-Transporting ATPases