THE BINDING OF RADIO‐IODINATED CALMODULIN TO PROTEINS ON DENATURING GELS

Calmodulin was iodinated by the method of Richman and Klee' with the following slight modifications. Iodination was performed (25°C) in a reaction volume of 1 ml that contained 0.05 M sodium phosphate (pH 7.01, 1 mg of calmodulin, 1 2 pg lactoperoxidase, and 0.4 mM CaCI,. Two mCi Na'"I were added to the reaction mixture and the reaction was initiated with 5 pl H,O, (diluted 1:500]. After 7 min, 5 pl of H,O, (1:500] was again added to the reaction mixture and the reaction was allowed to proceed for an additional 7 min. The iodinated calmodulin was isolated as described by Richman and Klee.' The specific activity of the calmodulin was approximately 1,000,000 cpm/pg (as determined by liquid scintillation counting). Calmodulin treated with chloramine-T has been shown to be inactivated.' and calrnodulin iodinated by this method is inactive in binding. We have not tried but believe calmodulin iodinated with the Bolton-Hunter reagent will also work, since it has been shown by others that calmodulin so treated can still activate cyclic nucleotide phosphodiesterase.