Identification of the Ca(2+)-dependent calmodulin-binding region of chromogranin A.
Chromogranin A (CGA), the most abundant protein in bovine adrenal chromaffin granules, is a high-capacity, low-affinity Ca(2+)-binding protein found in most neuroendocrine cells, and binds calmodulin (CaM) in a Ca(2+)-dependent manner. The binding of chromogranin A to calmodulin was determined by measuring the intrinsic tryptophan fluorescence of chromogranin A in the presence and absence of Ca2+. Binding was specifically Ca(2+)-dependent; neither Mg2+ nor Mn2+ could substitute for Ca2+. Chelation of Ca2+ by EGTA completely eliminated the chromogranin A-calmodulin interaction. CaM binding was demonstrated by a synthetic CGA peptide representing residues 40-65. When the CGA peptide and CaM were mixed in the presence of 15 mM CaCl2, the intrinsic tryptophan fluorescence emission underwent a substantial blue-shift, shifting from 350 to 330 nm. Like the intact CGA, the peptide-CaM binding was specifically Ca(2+)-dependent, and neither Mg2+ nor Mn2+ could induce the binding. Calmodulin bound both to CGA and to the synthetic CGA peptide with a stoichiometry of one to one. The dissociation constants (Kd) determined by fluorometric titration were 13 nM for the peptide-CaM binding and 17 nM for intact CGA-CaM binding. The Kd values are comparable to those (approximately 10(-9) M) of other CaM-binding proteins and peptides, demonstrating a tight binding of CaM by CGA. The CaM-binding CGA residues 40-65 are 100% conserved among all the sequenced CGAs in contrast to 50-60% conservation found in the entire sequence, implying essential roles of this region.
Yoo
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Laboratory of Cellular Biology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland 20892.
D000313:Adrenal Medulla / Q000737:chemistry / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000378:metabolism*; D002413:Cations, Divalent; D002417:Cattle; D002837:Chromaffin Granules / Q000737:chemistry; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D002147:Calmodulin; D002413:Cations, Divalent; D053379:Chromogranin A; D002864:Chromogranins; D010446:Peptide Fragments; D004533:Egtazic Acid; D014364:Tryptophan; D002118:Calcium