Isolation and characterization of calmodulin from spinach leaves and in vitro translation mixtures.

Calmodulin, a multifunctional calcium-modulated protein, has been isolated from spinach leaf tissue and from spinach leaf messenger RNA translation products. The translation protein and the spinach leaf protein have been partially characterized and compared to vertebrate calmodulins. Spinach leaf calmodulin will quantitatively activate bovine brain phosphodiesterase and will undergo a calcium-dependent shift in electrophoretic mobility similar to that of bovine brain calmodulin. In the presence of Ca(2+) the spinach and brain proteins comigrate, but in the presence of chelators they do not. A polyadenylylated RNA fraction has been isolated from spinach leaf tissue and translated in a wheat germ cell-free translation system. The calmodulin synthesized in vitro has been isolated by using calcium-dependent affinity chromatography on phenothiazine-Sepharose conjugates. The translation protein comigrates with spinach calmodulin during polyacrylamide gel electrophoresis whether in the presence or the absence of Ca(2+). The translation protein also undergoes a calcium-dependent mobility shift identical to that of spinach calmodulin. Amino acid analysis of the translation calmodulin indicates that it does not contain N(epsilon)-trimethyllysine, an amino acid residue that is characteristic of all calmodulins previously examined. These studies suggest that N(epsilon)-trimethyllysine is not required for the calcium-dependent interaction of calmodulin with phenothiazines and indicate the potential utility of phenothiazine-Sepharose conjugates as affinity-based adsorbents in biological and biochemical investigations.