Interaction of spinach leaf adenosine diphosphate glucose α-1,4-glucan α-4-glucosyl transferase and α-1,4-glucan, α-1,4-glucan-6-glycosyl transferase in synthesis of branched α-glucan

Chromatography of spinach leaf extracts on DEAE-cellulose resolves α-1,4-glucan, α-1,4-glucan 6-glycosyl transferase (branching enzyme) into two fractions. Branching enzyme fraction I which contains 10–20% of the total activity recovered from the DEAE-cellulose column is associated with the ADP-glucose α-1,4-glucan α-4-glucosyl transferase (α-glucan synthetase) fraction III. Separation of the branching enzyme I activity from the α-glucan synthetase III activity was achieved only by chromatography on an ADP-hexanolamine-Sepharose 4B column. The properties of the two branching enzyme fractions were very similar with respect to their activities toward potato amylose, amylopectin, and amyloses prepared with either rabbit muscle phosphorylase or spinach leaf α-glucan synthetase. Incubation of potato amylose with branching enzyme or incubation of phosphorylase or α-glucan synthetase with branching enzyme resulted in formation of glucan products resistant to complete hydrolysis by β-amylase (37–50%) and α-amylase (20–31%) but still quantitatively hydrolyzed by the combined action of pullulanase plus β-amylase. The only significant difference noted between the two branching enzyme activities is that fraction II has higher activity in citrate buffer than in other buffers in the pH range of 5.5–6.5 while fraction I has higher activity in bicine and phosphate buffers than with citrate buffer. Both branching enzyme fractions stimulate the previously described “unprimed activity” catalyzed by α-glucan synthetase fraction III about 11- to 14-fold. However, branching enzyme did not stimulate the primed activity. It was previously shown that the “unprimed reaction” was highly dependent on the presence of citrate and other anions. However, the Km of amylopectin and glycogen for α-glucan synthetase III which was found to be 0.53 mg/ml and 2.94 mg/ml, respectively, are decreased to 1.9 μ/ml and 0.86 μ/ml, respectively, in the presence of 0.5 m citrate. The levels at which primer are effective in the presence of 0.5 m citrate suggest that the “unprimed” synthetic activity previously reported may be due to the ability of the endogenous glucan associated with the α-glucan synthetase fraction to act as a primer when citrate or other anions are present. The stimulation of the “unprimed” activity by branching enzyme may then be explained by its catalysis of formation of an increased number of nonreducing chain ends in the growing glucan that are able to accept glucosyl residues from ADPglucose.