The C-terminal domain of the plasma membrane Ca2+ pump contains three high affinity Ca2+ binding sites.
The C-terminal portion of the plasma membrane Ca(2+)-ATPase contains different regulatory domains. A recombinant C-terminal fragment of the human plasma membrane Ca(2+)-ATPase 1b isoform (E1079-P1180) was used to study the role of two acidic amino acid stretches located on either side of the calmodulin binding domain, corresponding to synthetic peptides A18 (Vorherr, T., James, P., Krebs, J., Enyedi, A., McCormick, D. J., Penniston, J. T., and Carafoli, E. (1990) Biochemistry 29, 355-365) and B28 (James, P., Pruschy, M., Vorherr, T., Penniston, J. T., and Carafoli, E. (1989) Biochemistry 28, 4253-4258), respectively. The molecular mass of the recombinant C-terminal fragment, as determined by electrospray ionization mass spectrometry, was higher by 39 mass units than the calculated value (12,055 Da). This difference was the result of an EGTA-insensitive Ca2+ ion, which was located by chymotryptic proteolysis in a fragment corresponding to the last 37 amino acids of the expressed protein. Fluorescence experiments on the dansylated recombinant C-terminal fragment titrated with increasing amounts of free Ca2+ revealed two additional Ca2+ binding sites with affinities corresponding to KD values of about 30 and 300 nM, respectively. Stains All spectra of different synthetic peptides, corresponding to subdomains of the expressed protein, indicated that the site with the KD of 30 nM was probably located in the acidic sequence on the N-terminal side of the calmodulin binding domain (peptide A18); the site with the 300 nM KD was apparently located on the C-terminal side of the calmodulin binding domain (peptide B28) or, alternatively, formed by the cooperation of distant residues of the domain.
Institute of Biochemistry, Swiss Federal Institute of Technology (ETH), Zürich.
F
F
Hofmann
P
P
James
T
T
Vorherr
E
E
Carafoli
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D002462:Cell Membrane / Q000201:enzymology / Q000502:physiology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D007527:Isoenzymes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D000252:Calcium-Transporting ATPases; D002118:Calcium