Selective measurement of starch synthesizing enzymes in permeabilized potato tuber slices

Osmotically permeabilized potato (Solanum tuberosum L.) tuber slices were used to study the biosynthesis of starch under semi in vivo conditions. Criteria to distinguish the various enzymes involved in starch biosynthesis were developed based on the characteristics of the enzymes in in vitro experiments. Branching enzyme activity was inhibited at pH 8.5 or higher, while the starch synthases functioned optimally between pH 8.8 and 9.1. Unprimed soluble starch synthase activity was only apparent in the presence of sodium citrate (0.4 molar or higher). Granulebound and primed soluble starch synthase were active in the absence of sodium citrate. Primed soluble starch synthase activity was susceptible to inhibition by 10 millimolar zinc sulfate, while granule-bound starch synthase activity was not. The incorporation of the Glc moiety of ADP-Glc into starch in tissue slices by the various starch synthases was consistent with in vitro data with respect to the affinity of the enzymes for substrate, the pH profile, the stimulation by citrate, and the inhibition by zinc sulfate. These data were used to determine the activity of each of the starch synthases in tissue slices: granule-bound and soluble starch synthase transferred 37 and 55 picomoles ADP-Glc per hour per milligram fresh weight into starch of permeabilized tissue slices at 30°C and pH 9.1. In the presence of 0.5 molar sodium citrate, at least 40 picomoles ADP-Glc per hour per milligram fresh weight as transferred into starch by unprimed soluble starch synthase activity.