Polyclonal antibody that recognizes calcium-dependent determinants in Tetrahymena calmodulin

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Abstract
A precipitating antibody prepared against Tetrahymena pyriformis calmodulin recognizes Ca-dependent determinants in the native protein. The ability of the antibody to precipitate 35S-labeled Tetrahymena calmodulin in direct radioimmunoassays was enhanced at least 3-fold in the presence of Ca. Competitive radioimmunoassay using homogeneous preparations of endogenously 35S-labeled Tetrahymena calmodulin and protein A-Sepharose-purified IgG demonstrated that this antibody preparation is specific for protozoan calmodulin. Homogeneous vertebrate, invertebrate and plant calmodulins, as well as rabbit skeletal muscle troponin C, did not show significant competition with the 35S-labeled Tetrahymena protein at concentrations of 100-fold greater than that at which the homologous unlabeled Tetrahymena calmodulin produced 50% competition. A cyanogen bromide digest of tetrahymena calmodulin also showed partial competition with the intact 35S-labeled protein, but only in the presence of Ca. The major antigenic determinants were localized to the carboxyl-terminal half of the molecule by immunoassay of limited trypsin fragments of Tetrahymena calmodulin. The antibody bound native calmodulin complexed to bovine brain phosphodiesterase (EC 3.1.4.17) but failed to recognize the Tetrahymena calmodulin carboxyl-terminal fragment (76-147) when complexed to the enzyme.