Comparison of the Unit-chain Distributions of Glycogens from Different Biological Sources,Revealed by Anion Exchange Chromatography

The precise distributions of α-1,4-unit-chains of several kinds of glycogens, average chain length 7–13, from different sources, e.g., mammals, shellfish, mushrooms, and microorganisms, were compared using high performance anion exchange chromatography (HPAEC). These glycogens were completely debranched, and a series of maltosaccharides derived from individual unit-chains were analyzed. Most glycogens had similar chromatographic profiles, with degrees of polymerization (DPs), from 4–5 to 35; oyster glycogen contained shorter chains of DP 2–3. On the other hand, molar-based distribution patterns of these glycogens appeared to be specific to their origins. Ratios of their A- to B-chains varied from 0.6: 1 to 1.2: 1, as estimated by quantitative HPAEC of maltosaccharides by debranching of the β-limit dextrins. Possible chain lengths of the exterior A-chains were also discussed.     

Analyses of isoamylase gene activity in wild-type barley indicate its involvement in starch synthesis

The notion of debranching enzyme activity as a participant in starch synthesis is gaining acceptance. Inconsistent reports from mutant analyses implicate either isoamylase or pullulanase as a determinant in amylopectin formation and whether wild-type plants utilize one or the other, or both, of these debranching enzymes in starch synthesis is unclear. Recent results on the su1 mutant in maize suggest that both forms of debranching enzymes might be involved in amylopectin formation. We wished to find out if isoamylase takes part in starch synthesis by comparing isoamylase gene activity under three conditions: (1) during starch accumulation in developing sink tissues; (2) during starch degradation in germinating seeds; (3) in ectopic expression after applying sucrose, a starch precursor. We isolated the gene for barley isoamylase, iso1, and analysed its expression and regulation in germinating seeds, developing endosperm and vegetative tissues, and compared the isoamylase gene expression in sink tissues from three different species. Our results indicate that isoamylase gene activity is involved in starch synthesis in wild-type plants and is modulated by sucrose.     

The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.     

Starch- and glycogen-debranching and branching enzymes: Prediction of structural features of the catalytic (β/α)8-barrel domain and evolutionary relationship to other amylolytic enzymes

Sequence alignment and structure prediction are used to locate catalytic α-amylase-type (β/α)₈-barrel domains and the positions of their β-strands and α-helices in isoamylase, pullulanase, neopullulanase, α-amylase-pullulanase, dextran glucosidase, branching enzyme, and glycogen branching enzymes—all enzymes involved in hydrolysis or synthesis of α-1,6-glucosidic linkages in starch and related polysaccharides. This has allowed identification of the transferase active site of the glycogen debranching enzyme and the locations of β ? α loops making up the active sites of all enzymes studied. Activity and specificity of the enzymes are discussed in terms of conserved amino acid residues and loop variations. An evolutionary distance tree of 47 amylolytic and related enzymes is built on 37 residues representing the four best conserved β-strands of the barrel. It exhibits clusters of enzymes close in specificity, with the branching and glycogen debranching enzymes being the most distantly related.     

Action of Pseudomonas isoamylase on various branched oligo- and poly-saccharides

Pseudomonas isoamylase (EC 3.2.1.68) hydrolyzes (1 → 6)-α-D-glucosidic linkages of amylopectin, glycogen, and various branched dextrins and oligosaccharides. The detailed structural requirements for the substrate are examined qualitatively and quantitatively in this paper, in comparison with the pullulanase of Klebsiella aerogenes. As with pullulanase. Ps. isoamylase is unable to cleave D-glucosyl stubs from branched saccharides. Ps. isoamylase differs from pullulanase in the following characteristics: (1) The favored substrates for Ps. isoamylase are higher-molecular-weight polysaccharides. Most of the branched oligosaccharides examined were hydrolyzed at a lower rate, 10% or less of the rate of hydrolysis of amylopectin. (2) Maltosyl branches are hydrolyzed off by Ps. isoamylase very slowly in comparison with maltotriosyl branches. (3)Pr. isoamylase requires a minimum of three D-glucose residues in the B- or C-chain.     

Crystallisation of malto-oligosaccharides as models of the crystalline forms of starch: minimum chain-length requirement for the formation of double helices

The multigram preparation of malto-oligosaccharides of average d.p. ∼11, by the debranching of glycogen using Cytophaga isoamylase is described. Debranched glycogen and fractions derived therefrom readily crystallise from hot, concentrated aqueous solution to give 40–70% of crystalline materials having sharp X-ray diffraction patterns characteristic of A-, B-, and C-type (intermediate) starch polymorphs. The polymorphic form obtained is dependent on chain length, concentration, and temperature, the A-type being favoured by shorter chain-length, higher concentration, and higher crystallisation temperature. For pure oligomers, the minimum chain-length required for crystallisation (formation of double helices) is 10. In the presence of longer chains, oligomers as short as maltohexaose can co-crystallise. These results explain the known differences in aggregation properties of glycogens and amylopectins.     

The limit dextrinases from ungerminated oats (Avena sativa L.) and ungerminated rice (Oryza sativa L.).

The limit dextrinases from ungerminated oats and rice have been purified, and their substrate specificity compared with a bacterial isoamylase preparation. Both cereal enzymes could hydrolyse (1 yields6)-alpha-D-glucosidic linkages in oligosaccharide alpha-dextrins, pullulan, amylopectin, and the beta-limit dextrins of amylopectin and glycogen. However, under comparable conditions, they were unable to attack glycogens.     

Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15

The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned. Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase. The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes. These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes. Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase. The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase.     

The fine structure of the branched α-D-glucan from the blue-green alga Anacystis nidulans: comparison with other bacterial glycogens and phytoglycogen

The fine structure of the glycogen from the blue-green alga Anacystis nidulans has been examined. After selective hydrolysis of all (1→6)-α-D linkages by a bacterial isoamylase, the resulting mixture of linear chains was subjected to gel-permeation chromatography. For purposes of comparison, the glycogens from Escherichia coli and Arthrobacter sp., amylopectin, phytoglycogen from sweet corn, and shell-fish glycogen were treated similarly. The profiles of the unit chains of A. nidulans glycogen and phytoglycogen were closely similar. There was no close resemblance in the size distribution of unit chains for A. nidulans glycogen, other bacterial glycogens, and amylopectin.     

Starch debranching enzyme (R-enzyme or pullulanase) from developing rice endosperm: purification, cDNA and chromosomal localization of the gene

Starch debranching enzyme (R-enzyme or pullulanase) was purified to homogeneity from developing endosperm of rice (Oryza sativa L. cv. Fujihikari) using a variety of high-performance liquid chromatography columns, and characterized. A cDNA clone encoding the full length of the rice endosperm debranching enzyme was isolated and its nucleotide sequence was determined. The cDNA contains an open reading frame of 2958 bp. The mature debranching enzyme of rice appears to be composed of 912 amino acids with a predicted relative molecular mass (M_r) of 102069 Da, similar in size to its M_r of about 100 000 Da estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The amino acid sequence of rice debranching enzyme is substantially similar to that of bacterial pullulanase, while it bears little similarity to that of bacterial isoamylase or to glycogen debranching enzymes from human muscle and rabbit muscle. Southern blot analyses strongly suggest that the debranching enzyme gene is present as a single copy in the rice genome. Analysis by restriction fragment length polymorphism with a probe including the 3′-untranslated region of cDNA for rice debranching enzyme confirmed that the debranching enzyme gene is located on chromosome 4.     

Fine structural features of oyster glycogen: mode of multiple branching

The fine structural features of oyster glycogen, especially its mode of multiple branching, was investigated by repeated enzymic treatment with β-amylase and pullulanase, followed by the precise analysis of the -1,4-linked glucosyl unit-chains by high performance anion exchange chromatography (HPAEC). The purified glycogen (average mol. wt 8.5 × 10⁵, 11) obtained by DMSO-extraction from fresh oysters (Crassostrea gigas) collected in February (a time when the oysters are edible) showed a distribution of -1,4- -glucosyl unit-chains, with degrees of polymerization (dp) in the range 2–35 (dp 6, dominant), as measured by HPAEC after complete enzymic debranching. The oyster glycogen was subjected to stepwise degradations with β-amylase and pullulanase, and this procedure was repeated until complete hydrolysis was achieved (extent and degradation of 98% after five treatments). The yield of the limit dextrin formed at each trimming step and quantitative analysis of the unit-chain distributions indicated that the oyster glycogen has a highly branched structure (A:B-chain, 0.7:1), involving five or six times interlinkings of the chains (B-chains). Assuming that B1 chain carrying only A-chains, attaches by -1,6-bonds to another B-chain (B2 chain), which in turn attaches to a B3-chain, and so on, the molar ratios of the unit-chains (A, B1, B2-) of the dextrins during successive enzymic trimming showed that the ratio of A:B1:B2:B3:B4:B5-chain was 34:25:11:5:5:1, confirming the multiple ramified molecule. In connection with the digestion of oyster glycogen in the mammalian digestive tract, the glycogen was hydrolyzed by salivary and pancreatic -amylase, and several branched maltosaccharides in the digestion product were fractionated, and their structures determined using HPAEC.     

Synthesis of N-Carbamyl-d-2-thienyl-glycine and d-2-Thienylglycine by Microbial Hydantoinase

A debranching enzyme purified from germinating rice endosperm hydrolyzed oligosaccharides having maltosyl or maltotriosyl branches (B₄-B₆) moderately. Hydrolysis of maltosylmaltose by a “pullulanase” of higher plant origin has been scarcely reported, while our enzyme debranched maltosylmaltose like microbial pullulanase. Additionally, the enzyme slowly hydrolyzed isopanose to glucose and maltose.

Gel-filtration analyses of hydrolysis products of polysaccharides with the enzyme suggested that while it hydrolyzed α-1,6-linkages of pullulan at random, it hydrolyzed amylopectin and glycogen at the outer α-1,6-linkages preferentially In the hydrolysis products of glycogen with the enzyme for a longer incubation time, large molecular-weight glucans still remained. This indicated that the enzyme was able to hydrolyze a few of the α-1,6-linkages of glycogen.     

Synthesis of phenyl 2-acetamido-2-deoxy-4-O-α-l-fucopyranosyl-β-d-glucopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-6-O-α-l-fucopyranosyl-β-d-glucopyranoside

Maize and potato amylopectin (57 and 64%, respectively) were recovered as non-cyclic products from 4-h digests of the starches with cyclodextrin glycosyltransferase {(1→4)-α-d-glucan:[(1→4)-α-d-glucopyranosyl]transferase (cyclising), EC 2.4.1.19} from Klebsiella pneumoniae M 5 al. Besides smaller saccharides, highly branched fragments of different sizes (average d.p. 40–140) were obtained by fractionation. The extents of beta-amylolysis varied between 24 and 37%, indicating that the clusters were not equally susceptible to attack by cyclodextrin glycosyltransferase. The fragments of potato amylopectin still contained larger amounts of material of high molecular weight. Accordingly, part of the longer B-chains of the basic structure were protected from the enzymic attack, presumably because of interchain branches. By debranching with pullulanase, it was evident that the beta-limit dextrins of the fragments of potato amylopectin were composed of longer B-chains (average chainlength 17.8) than those of maize amylopectin (average chain-length 14.1). The A/B-chain ratios, which were calculated from h.p.l.c. data for the debranched beta-limit dextrins, were 1.22 (maize) and 1.06 (potato). Some structural differences between potato and maize amylopectin are discussed.     

Mutants of Arabidopsis lacking a chloroplastic isoamylase accumulate phytoglycogen and an abnormal form of amylopectin

Mutant lines defective for each of the four starch debranching enzyme (DBE) genes (AtISA1, AtISA2, AtISA3, and AtPU1) detected in the nuclear genome of Arabidopsis (Arabidopsis thaliana) were produced and analyzed. Our results indicate that both AtISA1 and AtISA2 are required for the production of a functional isoamylase-type of DBE named Iso1, the major isoamylase activity found in leaves. The absence of Iso1 leads to an 80% decrease in the starch content in both lines and to the accumulation of water-soluble polysaccharides whose structure is similar to glycogen. In addition, the residual amylopectin structure in the corresponding mutant lines displays a strong modification when compared to the wild type, suggesting a direct, rather than an indirect, function of Iso1 during the synthesis of amylopectin. Mutant lines carrying a defect in AtISA3 display a strong starch-excess phenotype at the end of both the light and the dark phases accompanied by a small modification of the amylopectin structure. This result suggests that this isoamylase-type of DBE plays a major role during starch mobilization. The analysis of the Atpu1 single-mutant lines did not lead to a distinctive phenotype. However, Atisa2/Atpu1 double-mutant lines display a 92% decrease in starch content. This suggests that the function of pullulanase partly overlaps that of Iso1, although its implication remains negligible when Iso1 is present within the cell.     

Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm

This is the first report on regulation of the isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5-12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total alpha-polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of isoamylase activity markedly lowered the gelatinization temperature from 54 to 43 degrees C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.     

A comparative study of edible canna (Canna edulis) starch from different cultivars. Part II. Molecular structure of amylose and amylopectin

Molecular structures of starches isolated from Japanese-green, Thai-green and Thai-purple cultivars of edible canna (Canna edulis Ker) were investigated. The absolute amylose content ranged from 19 to 25%. Degrees of polymerization (DPn) values of amylose determined by fluorescence-labeling method were 1590 for Thai-purple, 1620 for Japanese-green and 1650 for Thai-green cultivars. Mole% of branched fraction of amyloses from edible canna starches examined by a HPLC system after β-amylolysis of labeled amyloses was 13–16%. Branch chain-length distributions of amylopectin analyzed by HPSEC after debranching with isoamylase, followed by fluorescence-labeling of unit chain, showed bimodal distribution with the DPn range of 25–28. The amylopectin of edible canna starches contained high amounts of organic phosphorus (391–420 ppm). The distribution profile of phosphorylated chains, separated from non-phosphorylated chains by DEAE-Sephadex A-50 chromatography, indicated that the phosphate groups were located mostly in long B-chains of amylopectin molecules.     

Pullulanase in mung bean cotyledons. Purification, some properties and developmental pattern during and following germination

Starch debranching enzyme was purified from mung bean ( Vigna radiata ) cotyledons to investigate its properties and developmental pattern during and following germination. A debranching enzyme was purified up to the step where only a doublet of polypeptides with molecular masses of 99 and 101 kDa, respectively, was detected by SDS-PAGE. The enzyme is thought to be a single chain monomer, as the molecular mass of the enzyme determined by gel filtration was 72 kDa. Monoclonal antibodies raised against the purified preparation recognized the doublet, indicating that the two polypeptides have immunological homology to each other. The enzyme preparation showed a high activity with pullulan as a substrate, low activity with soluble starch and amylopectin, and no activity with glycogen. These substrate specificities indicate that the debranching enzyme from mung bean cotyledons is of the pullulanase type. Immunoblotting profiles revealed that the enzyme is present in dry seeds and decreases gradually after imbibition, suggesting the possibility that the pullulanase plays a role in developing mung bean cotyledons.     

Heterogeneity in branching of amylopectin

The angular dependence of scattered light from amylopectin and its β-limit dextrin, the mean square radius of gyration and the molecular weights M_w and M_n have been calculated on the basis of the cascade branching theory for the homogeneously branched model by Meyer &; Bernfeld (1940) (Model I) and for the two heterogeneously branched structures suggested by French (1972) (Model II) and by Robin et al. (1974, 1975) (Model III). The calculations take into account the particularities of topology in branched molecules and the experimentally determined ratio of the number of A- and B-chains, A/B = 1. Furthermore, an average branching density of 4% and an interconnecting chain length of ovbar|n_i2 = 22, found by gel permeation chromatography (GPC) after debranching, were used. The constraints lead to the conclusion that amylopectin is heterogeneously branched. Densely branched clusters containing 3·22 branching units are interconnected by longer chains of 22 units in length. Comparison of the calculated angular dependence of light scattering with measurements from a maize amylopectin β-limit dextrin in 1 n NaOH solution gives strong evidence for a modified Robin-Mercier model. The modification consists of the conclusion that the interconnecting chains are preferentially B-chains, such that these chains carry on the average 1·4 clusters, while Robin and Mercier assume exactly 2 clusters. Our result is in agreement with the distribution of chain length found after debranching the amylopectin β-limit dextrin.     

The effects of amylose content on the molecular size of amylose, and on the distribution of amylopectin chain length in maize starches

The amylose to amylopectin ratios in six maize starch samples of differing amylose contents were measured by enzymatic debranching, followed by high performance size exclusion chromatography (HPSEC). The molecular size of amyloses, estimated by -log K_wav, shows progressive decrease with the increase in amylose content in maize starches. The gel permeation chromatographs of the corresponding amylopectins, debranched with isoamylase, showed bimodal distributions containing long and short chains. The average chain length of amylopectin has a correlation with amylose content. The correlation coefficients between amylose content and average chain length, long chain length, weight ratio and the mole ratio of long and short chain length, were 0.97, 0.92, 0.96, 0.94 respectively. The maize starch with the highest amylose content has the lowest amylose molecular size and the longest chains, with a high ratio of long to short chains in its amylopectin fraction. Comparing the values of amylose content determined by HPSEC of starch or debranched starch with those of the iodinecomplex method, we conclude that long chains of amylopectin in high amylose starches contribute significantly to apparent amylose content.     

Towards a better understanding of the metabolic system for amylopectin biosynthesis in plants: rice endosperm as a model tissue

Starch is made up of amylose (linear alpha-1,4-polyglucans) and amylopectin (alpha-1,6-branched polyglucans). Amylopectin has a distinct fine structure called multiple cluster structure and is synthesized by multiple subunits or isoforms of four classes of enzymes: ADPglucose pyrophosphorylase, soluble starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE). In the present paper, based on analyses of mutants and transgenic lines of rice in which each enzyme activity is affected, the contribution of the individual isoform to the fine structure of amylopectin in rice endosperm is evaluated, and a new model referred to as the "two-step branching and improper branch clearing model" is proposed to explain how amylopectin is synthesized. The model emphasizes that two sets of reactions, alpha-1,6-branch formation and the subsequent alpha-1,4-chain elongation, are catalyzed by distinct BE and SS isoforms, respectively, are fundamental to the construction of the cluster structure. The model also assesses the role of DBE, namely isoamylase or in addition pullulanase, to remove unnecessary alpha-1,6-glucosidic linkages that are occasionally formed at improper positions apart from two densely branched regions of the cluster.