Elucidation of a minimal immunoreactive site of vertebrate calmodulin.
The heptapeptide Asn-Tyr-Glu-Glu-Phe-Val-Gln-NH2 corresponding to residues 137-143 of vertebrate calmodulin is as immunoreactive as the entire 148-residue protein. A reproducible and rapid procedure for producing antisera against vertebrate calmodulin has been previously described (L. J. Van Eldik and D. M. Watterson (1981) J. Biol. Chem. 256, 4205-4210). Most of the antisera elicited by this method react with a major immunoreactive region (residues 127-144) in the COOH-terminal domain of vertebrate calmodulin. In this report, the minimum segment of calmodulin required for reactivity with an antiserum that readily distinguishes various types of calmodulins is defined. These studies demonstrate that a linear segment of seven amino acid residues shows a competition curve in radioimmunoassay resembling the competition curve of intact calmodulin. This heptapeptide is the smallest calmodulin segment and the only seven-residue segment in the 135-145 region that shows quantitative immunoreactivity with the anti-calmodulin serum. These data demonstrate that this heptapeptide is a major immunoreactive site of calmodulin. However, when this immunoreactive site heptapeptide is conjugated to a carrier and injected into rabbits, it does not elicit antisera that react with the native protein. These studies demonstrate that quantitative immunoreactivity of antisera produced in animals can be found in small peptide segments and that, for calmodulin, the requirements for production of anti-peptide antibodies that react with the native protein molecule are not as simple as surface exposure of the peptide region.
D000818:Animals; D000917:Antibody Formation; D001665:Binding Sites; D002147:Calmodulin / Q000276:immunology*; D002645:Chickens; D005260:Female; D005895:Gizzard, Avian / Q000378:metabolism; D007120:Immunochemistry; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification; D011817:Rabbits; D011863:Radioimmunoassay
D002147:Calmodulin; D010446:Peptide Fragments