Photoaffinity labeling study of the interaction of calmodulin with the plasma membrane Ca2+ pump.
Bovine brain calmodulin was labeled with synthetic peptides corresponding to the calmodulin-binding domain of the erythrocyte plasma membrane Ca(2+)-ATPase. One 20-amino acid peptide and two 28-amino acid peptides were used, carrying L-4'-(1-azi-2,2,2-trifluoroethyl)phenylalanine residues in position 9 (peptides C20W* and C28W*) and position 25 (peptide C28WC*), respectively. The localization of the contact regions between calmodulin and the N- and C-terminal portions of the peptides was the aim of this study. The three peptides were N-terminally blocked with a 3H-labeled acetyl group to facilitate the identification of labeled fragments after isolation and digestion. The binding site for phenylalanine 25 was identified in the N-terminal domain of calmodulin while the phenylalanine derivative in position 9 labeled the C-terminal domain. Fluorescence studies using the dansylated N- and C-terminal halves of calmodulin and peptide C20W corresponding to the first 20 amino acids of the calmodulin-binding domain showed that only the C-terminal lobe of calmodulin had high affinity for the peptide (KD in the nanomolar range).
Vorherr
T
T
Laboratory of Biochemistry, Swiss Federal Institute of Technology (ETH), Zurich.
Quadroni
M
M
Krebs
J
J
Carafoli
E
E
D000345:Affinity Labels / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000097:blood / Q000378:metabolism* / Q000502:physiology*; D002147:Calmodulin / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D010450:Endopeptidases; D004910:Erythrocyte Membrane / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism
D000345:Affinity Labels; D002147:Calmodulin; D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D010450:Endopeptidases; D000252:Calcium-Transporting ATPases; D003488:Cyanogen Bromide