Calmodulin resolution of multiple peaks of activity by preparative electrofocusing.

When the supernatant fractions from rat brain homogenates were subjected to preparative electrofocusing in a bed of Sephadex G75, several peaks of calmodulin were resolved. A minor peak representing free calmodulin migrated with a pI of 3.8 --4.4. Other peaks of calmodulin activity were observed with isoelectric points at pH 4.8, 5.2, 6.0 and 6.8. The peak of calmodulin activity at 5.2 co-migrated with phosphodiesterase activity which was stimulated 1.8-fold by calcium. A second peak of phosphodiesterase activity detected at pH 8.0 was stimulated 1.2-fold by calcium and occurred in an area where no calmodulin activity could be detected. If isoelectric focusing was done in the presence of 8 M urea only one peak of calmodulin activity was observed with a pI of 4.0--4.4. It is suggested that the multiple peaks of calmodulin resolved by electrofocusing represent calmodulin associated with various proteins which are subject to modulation by calmodulin and calcium.