Amylopectin enzymic analysis

This enzymic analysis for the numbers of A chain binding to a Ba chain provides basic information for elucidation and characterizaticxi of the intricate branching of amylopectin. 

Wong, K. S., Jane, J. -L. (1997). Quantitative analysis of debranched amylopectin by HPAEC-PAD with a postcolumn enzyme reactor. Journal of Liquid Chromatography, 20,297-310. 

An additional problem in limit-dextrin analysis requires comment. Assuming (a) complete phosphorolysis and /3-amylolysis and (b) the presence of equal numbers of A- and B-chains, the difference A between the CL of a d>-dextrin and a S-dextrin of various polysaccharides should be constant. This is not the case. With rabbit-liver glycogen, wheat amylopectin, and maize amylopectin, A was 1.8, 2.6, and 2.7 n-glucose residues, and for seventeen other polysaccharides, A varied between 1.1 and 2.6. If the ( -dextrin had single-unit A-chains, this variation could be ascribed to differences in the ratio of A-chains to B-chains. This conclusion is probably incorrect, and no ready explanation for the variation in A is available as, in all the experiments, enzymic degradation was essentially complete. 

In addition to its in vivo importance, R-enzyme is extremely useful for structural analysis. For example, the release of maltose and maltotriose from amylopectin jS-dextrin by R-enzyme provides good evidence of multiple branching, since the yield (12.8%) is near to that calculated (10.4%) for a tree-type structure having equal numbers of A- and B-chains. In addition, the simultaneous action of R-enzyme and 8-amylase can be used for the determination of CL (for details, see Ref. 144). 

Use of MALDI-TOF-MS allows relatively straightforward separation and identification of oligosaccharides with DPs up to 30. This feature makes it possible to identify products from enzymatic debranching of amylopectin. Richardsson et al. employed MALDI-TOF-MS and ESI-MS for enzyme-aided structure analysis of cationic potato amylopectin. " By MALDI-TOF-MS, identification of the oligosaccharides with DP 6-20 obtained from hydrolysis with pullulanase could be performed. Use of ESI-MS", on the other hand, provided detailed information on the chemical structure of oligosaccharides with DP <5. However, the lack of suitable standards prevented quantification by MALDI-TOF-MS and ESI-MS. 

The subcellular localization of the starch biosynthetic and degradative enzymes of spinach leaves was carried out by measuring the distribution of the enzymes in a crude chloroplast pellet and in separated components of a protoplast lysate. The enzymes which were involved in the syntheses of starch were detected in the chloroplasts, whereas some of the enzymes involved in the degradation of starch were mainly in the soluble protein fraction but were also found in the chloroplast. The digestion pattern of the amylase on amylopectin as substrate indicated that the enzyme was an a-amylase from its endo-lytic activity, but displayed properties unlike the typical a-amylase isolated from endosperm tissue. A time-sequence analysis of the starch digestion pattern in germinating... 

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