Starch biosynthesis: further evidence against the primer nonreducing-end mechanism and evidence for the reducing-end two-site insertion mechanism

Two reactions were studied with three varieties of starch granules from maize, wheat, and rice. In Reaction-I, the granules were reacted with 1 mM ADP-[14C]Glc and in Reaction-II, a portion of the granules from Reaction-I was reacted with 1 mM ADP-Glc. The starch granules were solubilized and reacted with the exo-acting glucoamylase and beta-amylase to an extent of 50% or less of the 14C-label. The amounts of 14C-labeled products from glucoamylase and beta-amylase were nearly equal for Reaction-I and Reaction-II. If the addition had been to the nonreducing ends of primers, Reaction-II would not have given any labeled products from the hydrolysis of glucoamylase and beta-amylase. These results indicate that the elongation of the starch chain is the addition of D-glucose to the reducing end by a de novo two-site insertion mechanism and not by the addition of D-glucose to the nonreducing end of a primer. This is in conformity with previous results in which starch granules were pulsed with ADP-[14C]Glc and chased with nonlabeled ADP-Glc, giving 14C-labeled D-glucitol from the pulsed starch and a significant decrease in 14C-labeled D-glucitol from the chased starch on reducing with NaBH4 and hydrolyzing with glucoamylase [Carbohydr. Res. 2002, 337, 1015–1022]. It also is in conformity with the inhibition of starch synthesis that occurs when putative primers are added to starch granule–ADP-Glc digests, indicating that the elongation is not by the nonreducing-end primer mechanism [Carbohydr. Res. 2005, 340, 245–255].