Determination of the starch-phosphorylating enzyme activity in plant extracts

For quantification of α-glucan, water dikinase (GWD) activity in crude extracts of plant tissues a radio-labeling assay was established that uses soluble starch and 33P-labeled ATP as phosphate acceptor and donor, respectively. A constant rate of starch labeling was observed only if the ATP applied was labeled at the β position. In wild-type extracts from leaves of Arabidopsis thaliana (L.) Heynh. the maximum rate of starch phosphorylation was approximately 27 pmol min-1 (mg protein)-1. Leaf extracts from the GWD-deficient sex1 mutants of Arabidopsis showed no significant incorporation of phosphate whereas extracts from potato (Solanum tuberosum L.) tuber expressing a GWD antisense construct exhibited less activity than the wild-type control. To our knowledge this is the first time that a quantification of the starch-phosphorylating activity has been achieved in plant crude extracts.