In vitro biosynthesis of phosphorylated starch in intact potato amyloplasts

Intact amyloplasts from potato (Solanum tuberosum L.) were used to study st arch biosynthesis and phosphorylation. Assessed by the degree of intactness and by the level of cytosolic and vacuolar contamination, the best prepara tions were selected by searching for amyloplasts containing small starch gr ains. The isolated, small amyloplasts were 80% intact and were free from cy tosolic and vacuolar contamination. Biosynthetic studies of the amyloplasts showed that [1-C-14]glucose-6-phosphate (Glc-6-P) was an efficient precurs or for starch synthesis in a manner highly dependent on amyloplast integrit y. Starch biosynthesis from [1-C-14]Glc-1-P in small, intact amyloplasts wa s 5-fold lower and largely independent of amyloplast intactness. When [P-33 ]Glc-6-P was administered to the amyloplasts, radiophosphorylated starch wa s produced. isoamylase treatment of the starch followed by high-performance anion-exchange chromatography with pulsed amperometric detection revealed the separated phosphorylated alpha-glucans. Acid hydrolysis of the phosphor ylated alpha-glucans and high-performance anion-exchange chromatography ana lyses showed that the incorporated phosphate was preferentially positioned at C-6 of the Glc moiety. The incorporation of radiolabel from Glc-1-P into starch in preparations of amyloplasts containing large grains was independ ent of intactness and most likely catalyzed by starch phosphorylase bound t o naked starch grains.