Identification and characterization of the calmodulin-binding domain of neuromodulin, a neurospecific calmodulin-binding protein.
Neuromodulin (formerly designated P-57) is an abundant, neural specific, calmodulin-binding protein which exhibits higher affinity for calmodulin in the absence of free Ca2+ than in the presence of free Ca2+. In this study a series of proteolytic fragments of neuromodulin were systematically screened for calmodulin-Sepharose binding activity. A 9-amino acid fragment, designated M1-C1 and having the sequence RGHITRKKL, was identified as the putative CaM-binding domain of neuromodulin. Two heptadecapeptides, designated FP57-Phe and FP57-Trp, were synthesized, each containing the M1-C1 sequence and the four flanking amino acids from each site. The FP57-Trp peptide contained a tryptophan residue in place of the native phenylalanine. Anti-FP57-Phe antibody binding to neuromodulin was inhibited by preincubation of antibodies with excess FP57-Phe. 125I-CaM gel overlay of neuromodulin was inhibited by anti-FP57-Phe antibodies. Addition of CaM to FP57-Trp increased peptide tryptophanyl fluorescence. In the presence of Ca2+, the stoichiometry of the FP57-Trp.CaM complex was 1:1, FP57-Trp binding to CaM was competitive with neuromodulin. The Ca2+-independent dissociation constant of the FP57-Phe.CaM complex was 0.41 microM. The Ca2+-dependent affinity of the complex could not be measured directly but appeared to be significantly greater than the Ca2+-independent affinity.
Alexander
K A
KA
Department of Pharmacology, School of Medicine, University of Washington, Seattle 98195.
Wakim
B T
BT
Doyle
G S
GS
Walsh
K A
KA
Storm
D R
DR
D000595:Amino Acid Sequence; D001345:Autoradiography; D001665:Binding Sites; D001667:Binding, Competitive; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D002942:Circular Dichroism; D019922:GAP-43 Protein; D009682:Magnetic Resonance Spectroscopy; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments