Development of an assay for Mg-dechelatase of oilseed rape cotyledons, using chlorophyllin as the substrate

Chlorophyllin (Chlin), the Mg-chlorin obtained from chlorophyll (Chl) was employed as substrate of Mg-dechelatase. The release of Mg²⁺ was associated with a shift of absorption from 644 to 687 nm. Changes of absorption at 687 nm were taken as a measure of Mg-dechelatase activity present in preparations of oilseed rape thylakoids. Absorption changes were correlated linearly with enzyme dose. The pH optimum of Mg release from Chlin was ca 9 with a broad flank down to pH 7. The reaction showed saturation kinetics with an apparent K_m value of ca 17 n M . The activity was inhibited in the presence of cysteamine or reduced glutathion. There was no effect of the thiol reagent N-ethyl maleimide. The bulk of dechelatase activity was associated with the chloroplast membranes. The enzyme is partially latent and the appearance of full activity requires the solubilization of thylakoids with detergent. The highest activities were detected in mature green rape cotyledons. During dark-induced senescence the activity declined at roughly the same rate as Chl was lost in the leaf tissue.