Calmodulin inhibitor in senescing apples and its physiological and pharmacological significance

While assaying calmodulin activity in senesced apple extracts by using its property of promoting the activity of activator-deficient 3',5'-cyclic AMP 5'-nucleotidohydrolase (phosphodiesterase, EC 3.1.4.17) from bovine heart, we detected a heat-stable, dialyzable, low molecular weight component that inhibited calmodulin activity. Specific activity of calmodulin as calculated from the linearly increasing portion of the activity curve was in the range of 150 to 160 units/mg of protein in crude extracts from apples stored at 2 degrees C for a period of 6 months with or without calcium treatment. Apple extract that was passed through a phenothiazine-Sepharose affinity column did not promote phosphodiesterase activity, whereas the EGTA eluate of the column promoted phosphodiesterase activity similar to the original extract. The inhibition of calmodulin activity appeared to be lower in extracts from apples stored at 2 degrees C after calcium treatment. The inhibition was found to increase after storage of apples at room temperature for 30 days. Activity of purified bovine brain calmodulin was also inhibited by the inhibitor present in apple extracts, which indicated that the inhibition was not specific to plant calmodulin alone and could have wide applications. The importance of the inhibitor in relation to senescence/aging and its possible pharmacological applications are discussed.