Quantitative Assay Method for Starch Branching Enzyme with Bicinchoninic Acid by Measuring the Reducing Terminals of Glucans

Abstract

Starch is a glucose polymer with two α glucosidic linkages, and linearly linked α 1,4 glucosidic chains are branched by α 1,6 glucosidic linkages. Starch is composed of linear or fewly branched amylose and highly branched amylopectin. Amylopectin has a distinct highly ordered structure called "tandem cluster structure", in which most side chains are arranged in parallel and pairs of neighboring chains form double helices 1) when linear portions of these chains reach the length equivalent to degree of polymerization (DP) > 10. 2 5) The formation of double helices in the amylopectin cluster dramatically induces its hydrophobicity and crystallinity. These specific features of amylopectin fine structure are enabled by the localization of position of branches in the cluster. 6) The starch synthesis system has developed during the process of evolution of plants, 6,7) and key enzymes involved in the construction of amylopectin tandem cluster structure have differentiated into multiple isozymes with distinct functions whereas in glycogen synthesizing organisms such as bacteria and animals no such functional differentiations in glycogen synthesis enzymes have occurred. 8,9) Starch branching enzyme (BE) plays an important part in the formation of branches in amylopectin molecules. Green plants are known to have two types of BE isozymes, BEI and BEII. In addition, BEII is further differentiated into BEIIa and BEIIb isoforms in cereals although BEIIb is usually specifically expressed in endosperm while BEIIa is ubiquitously present in every tissue. 10 13) Our biochemical studies on three mutants of rice which are defective in BEI, BEIIa and BEIIb, respectively, strongly suggest that the role of BEIlb is most specific in synthesizing branches located on the basal portion of the crystal zone (referred to as the crystal lamellae) of the cluster because BEI and BEIIa can hardly complement its role in its absence. 8,14) On the other hand, BEI plays an important part in forming branches which are positioned at the basal part of the cluster in the less crystalline zone (referred to as the amorphous lamellae), and those which link the clusters, but its role can be largely complemented by BEIIb and BEIIa in the BEI mutant. 15) Although the activity of BEIIa accounts for about 20% of the total BE activity in rice endosperm, the specificity of its function must be poor because no significant changes in the structure of amylopectin or the physicochemical properties of starch granules are found in the BEIIa mutant (Nishi et al ., unpublished data).In vitro studies using purified BE isozymes revealed that BEI and BEII differ in terms of specificity for glucans, amylose or amylopectin, and the length of glucan chains transferred; BEI prefers to attack amylose to amlopectin and preferentially transfers various glucan chains with DP values greater than 6, whereas BEII prefers to use amylopectin to amylose, and preferentially transfers short chains with DP6 in addition to DP7. 16 23) In past BE investigations, the activity has been assaye...

Functional Diversity of Isoamylase Oligomers: The ISA1 Homo-Oligomer Is Essential for Amylopectin Biosynthesis in Rice Endosperm

“…The mixtures including different amounts of purified OsISA1 and/or OsISA2 proteins as above (E) were incubated in 100 mL of the buffer solution containing 50 mM MES-NaOH (pH 6.0) and 0.5 mg of rice amylopectin or rice phytoglycogen at 30°C for 10 min. DBE activity was determined by measuring the amount of reducing ends increased after the DBE reaction according to the method described by Utsumi et al (2009). The numbers on the horizontal axis correspond to the lanes in E. The activity was expressed as relative values when the activities of OsISA1 (2 mg) only toward amylopectin or phytoglycogen (lane 2) were estimated to be 100%.…”

Convergent Evolution of Polysaccharide Debranching Defines a Common Mechanism for Starch Accumulation in Cyanobacteria and Plants

“…The DBE activity was determined by measuring the amount of reducing ends in the reaction products according to the method of Utsumi et al (2009). Solution A consisted of 97.1 mg of disodium 2,2'-bicinchoninate, 3.2 g of sodium carbonate monohydrate, and 1.2 g of sodium bicarbonate in a total volume of 50 mL.…”

Structural features of a bacterial cyclic α-maltosyl-(1→6)-maltose (CMM) hydrolase critical for CMM recognition and hydrolysis

“…The mixture was incubated at 25°C for 6 min. After stopping the reaction by heat treatment at 100°C for 10 min, the reducing power of each mixture was measured by the bicinchoninic acid assay according to the method of Utsumi et al (58). For the kinetic analysis for MM, substrate solutions containing 1.25-75 mM MM were used.…”