Branching enzymes, assay

Assay procedures there are three accepted methods in assaying branching enzyme (BE). One measures the decrease in absorbance of the glucan-I3 complex that results from the branching of amylose or amylopectin. The assay mixture containing amylose or amylopectin is incubated and aliquots are taken and iodine reagent is added.35,247 The decrease of absorbance is measured at 660 nm for amylose and at 530 nm for amylopectin. 

Several assays (Fig. 1) are available to measure branching enzyme activity. The iodine assay is based on the decrease in absorbance of the glucan-iodine complex (Krisman, 1962) resulting from the branching of amylose or amylopectin by the enzyme, provided that a-amylases are absent from the enzyme preparation or their activity is greatly reduced by adjusting the assay conditions. 

SPECIFIC ACTIVITIES (UNITS/MG OF PROTEIN) OF BRANCHING ENZYME ISOFORMS AS MEASURED USING DIFFERENT ASSAY METHODS" ... 

It is worth noting that a conservative change from Glu to Asp resulted in higher specific activities in all three types of assays. It is interesting that all higher plant branching-enzymes have an Asp at the position occupied by Glu-459 in E. coli. 

In the second approach, herbicide-resistance mutations in the Arabidopsis ALS gene were studied in E. coli. To do this, wild type and mutant Arabidopsis genes were functionally expressed in E. coli, such that the plant genes complemented a branched chain amino acid auxotrophy in the bacteria (Smith et al. 1989, PNAS in press). ALS enzyme assays on extracts prepared from E. coli expressing the mutant Arabidopsis gene indicated that the mutant enzyme is resistant to sulfonylurea herbicides but is sensitive to the imidazolinone herbicide imazaquin. This selective... 

Branching enzyme is assayed by measurement of the decrease in optical density of the amylopectin-iodine complex at 570 mu, after incubation at 30° and pH 7. Plant Q-enzymes have no effect on amylopectin and thus differ significantly from the animal branching-enzjrmes. For this reason, it was suggested " that the term Q-enzyme be confined to plant branching-enzymes. 

The benzoquinones and naphthoquinones are derived from the shikimate pathway (Figure 2), which has been described as a metabolic tree with many branches. Most of the information concerning the biosynthesis of MK and Q was obtained with E. coli hy using isotopic tracers, by the isolation of mutants, and by the accumulation of intermediates and enzyme assays. Due to space limitations, only a general account is given here for more information, several comprehensive reviews should be consulted. ... 

Individual measurement of all five tryptophan pathway enzymes have been reported from tobacco and carrot cell cultures, wheat, corn, and peas (Widholm, 1973 Singh and Widholm, 1974 Hankinsa/, 1976). The relative amounts of the enzymes assayed in vitro differ with the various sources. However, Singh and Widholm (1974) reported extractable quantities of each enzyme in wheat, regardless of the tissue source or plant age, sufficient to synthesize the amount of tryptophan present within the same tissue in 48 h. No in vitro aggregation of any of the tryptophan branch enzymes was ob-... 

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