Binding of simple peptides, hormones, and neurotransmitters by calmodulin.
We have prepared a fluorescent conjugate of porcine calmodulin with 5-(dimethylamino)-1-naphthalene-sulfonyl chloride that is highly sensitive to both calcium binding and protein binding. We have used the fluorescence of this conjugate in addition to the intrinsic peptide fluorescence to show that adrenocorticotropic hormone (ACTH), beta-endorphin, glucagon, and substance P undergo calcium-dependent binding by calmodulin, with competition for common binding sites. The dissociation constants determined in the presence of 0.85 mM CaCl2 and 0.2 N KC1, pH 7.3 at 25 degrees C, range from 1.5 muM to 3.4 muM. The alpha-melanocyte-stimulating hormone, bombesin, and somatostatin also bind, with dissociation constants between 60 muM and 90 muM. Angiotensins I and III, bradykinin, neurotensin, physalaemin, substance P octapeptide, insulin, and Leu- and Met-enkephalin show little or no binding. Sequence comparisons show that the peptides that bind calmodulin well contain regions structurally similar to the recognition sequence for the cAMP-dependent protein kinase and to the sequences surrounding phosphorylated serine residues in several calmodulin binding proteins. This result suggests that modification of calmodulin binding sites in calmodulin-dependent proteins is one of the functions of protein kinase. Calcium has a dual role in peptide binding by calmodulin. The occupation of calcium binding sites having a pK approximately 4 results in a 2-fold increase in peptide binding affinity.
D000324:Adrenocorticotropic Hormone / Q000378:metabolism; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D004723:Endorphins / Q000378:metabolism; D005934:Glucagon / Q000378:metabolism; D006728:Hormones / Q000378:metabolism*; D018377:Neurotransmitter Agents / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013373:Substance P / Q000378:metabolism; D013552:Swine; D001615:beta-Endorphin
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D004723:Endorphins; D006728:Hormones; D018377:Neurotransmitter Agents; D010455:Peptides; D013373:Substance P; D001615:beta-Endorphin; D000324:Adrenocorticotropic Hormone; D005934:Glucagon