Identification of Naturally Occurring Calmodulin Inhibitors in Plants and their Effects on Calcium- and Calmodulin-promoted Protein Phosphorylation12

摘要

While studying the calmodulin activity in post-climacteric apples, a heat stable, dialyzable component that inhibited calmodulin-promoted phosphodiesterase activity was detected. The compound(s) that inhibited calmodulin activity did not bind to Dowex-50, H+or Dowex-2, Cl−and was exclusively present in the neutral fraction. The inhibitors irreversibly bound to polyvinylpolypyrrolidone indicating their phenolic nature. Fractionation of the neutral fraction on a C18-μbondapak column and analysis for the inhibition of calmodulin-promoted phosphodiesterase activity showed significant inhibitory activity associated with fractions eluted 5 min, 15 min and 18 min after injection. Perdeuteriomethylation and combined gas chromatographic-mass spectrometric analysis of the inhibitors showed them to be flavonoids. (+)-Catechin was identified in the fraction eluted 5 min after injection that also showed maximum inhibition. Other flavonoids such as epicatechin, quercetin and naringenin also inhibited calmodulin-promoted phosphodiesterase activity. Among the phenolic compounds commonly encountered in plant tissue only caffeic acid inhibited calmodulin-promoted phosphodiesterase activity. Inhibition by catechin nd caffeic acid could be reversed by increasing the calmodulin concentration in the assay mixture. Both catechin and caffeic acid inhibited Ca- and calmodulin-promoted phosphorylation of soluble proteins from corn coleoptiles. The physiological properties of flavonoids are discussed in light of this eviden