Isolation, purification and cell-free synthesis of calmodulin from the pig anterior pituitary gland.

Calmodulin was extracted and purified from pig anterior pituitary gland. The protein was characterized by its migration on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in the presence of Ca2+ or EGTA, its U.V. spectrum between 240 and 290 nm and the activation of calmodulin-deficient cyclic AMP phosphodiesterase. The yield was 370 mg/kg wet wt. mRNA was also extracted from the same tissue and translated in a wheat-germ cell-free translation system. Translated calmodulin was identified by its heat-stability, its co-migration with authentic anterior-pituitary calmodulin on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, its acidic isoelectric point (4.15) on flat-bed isoelectric focusing, its Ca2+-dependent binding to fluphenazine-Sepharose 6B, and its co-elution from this gel with authentic unlabelled calmodulin with EGTA buffer. Calmodulin was not translated as a precursor form. In this tissue it was calculated that calmodulin accounted for 0.5-1% of the total translated protein.