DEVELOPMENT OF AN ASSAY FOR MG-DECHELATASE OF OILSEED RAPE COTYLEDONS, USING CHLOROPHYLLIN AS THE SUBSTRATE

Chlorophyllin (Chlin), the Mg-chlorin obtained from chlorophyll (Chi) was employed as substrate of Mg-dechelatase. The release of Mg2+ was a ssociated with a shift of absorption from 644 to 687 nm. Changes of ab sorption at 687 nm were taken as a measure of Mg-dechelalase activity present in preparations of oilseed rape thylakoids. Absorption changes were correlated linearly with enzyme dose. The pH optimum of Mg relea se from Chlin was ca 9 with a broad flank down to pH 7. The reaction s howed saturation kinetics with an apparent K-m value of ca 17 nM. The activity was inhibited in the presence of cysteamine or reduced glutat hion. There was no effect of the thiol reagent N-ethyl maleimide. The bulk of dechelatase activity was associated with the chloroplast membr anes. The enzyme is partially latent and the appearance of full activi ty requires the solubilization of thylakoids with detergent. The highe st activities were detected in mature green rape cotyledons. During da rk-induced senescence the activity declined at roughly the same rare a s Chi was lost in the leaf tissue.