Structural, physicochemical, and pasting properties of starches from potato plants with repressed r1-gene.

The aim of this work was to investigate the effect on starch molecular and physicochemical properties of down regulation of the R1 protein in potato (Solanum tuberosum L. cv. "Dianella") tubers. Most prominent is a 90% suppression of the phosphate content in the isolated potato tuber starch. The amylopectin chain length distribution profile as determined by HPAEC/PAD was not affected, but the amylose content was increased in the most down-regulated plants. The pasting properties of the transgenic starch revealed a pronounced decrease in peak viscosity and increased setback viscosity as measured using a rapid Visco analyzer. The starch gels displayed an increased hardness and stickiness with a maximum at 1.7 nmol of Glc-6P mg-1 of starch compared to the control lines. At very low phosphate levels (1.4 nmol of Glc-6P mg-1 of starch), the gel hardness was decreased as a result of increased gel brittleness. The increase in gel brittleness is believed to be an effect of an increased proportion of free amylopectin blocklets in the starch as determined by SEC/RI. The possible links between the structural and physicochemical parameters are discussed.

The action of starch synthase II on 6"'-alpha-maltotriosyl-maltohexaose comprising the branch point of amylopectin.

The principle of using a chemically synthesized, well-defined branched oligosaccharide to provide a more detailed knowledge of the substrate specificity of starch synthase II (SSII) is demonstrated. The branched nonasaccharide, 6"'-alpha-maltotriosyl-maltohexaose, was investigated as a primer for particulate SSII using starch granules prepared from the low-amylose pea mutant lam as the enzyme source. The starch granule preparation from the lam pea mutant contains no starch synthases other than SSII and is devoid of alpha-amylase, beta-amylase and phosphorylase activity. SSII was demonstrated to catalyse a specific nonprocessive elongation of the nonreducing end of the shortest unit chain of 6"'-alpha-maltotriosyl-maltohexaose, i.e. the maltotriose chain. Maltotriose and maltohexaose, representing the two linear building units of the branched nonasaccharide, were also tested as primers for SSII. Maltotriose was elongated more efficiently than 6"'-alpha-maltotriosyl-maltohexaose and maltohexaose was used less efficiently. Compared to the surface exposed alpha-glucan chains of the granule bound amylopectin molecules, all three soluble oligosaccharides tested were poor primers for SSII. This indicates that in vivo, the soluble oligosaccharides supposedly released as result of amylopectin trimming reactions are not re-introduced into starch biosynthetic reactions via the action of the granule bound fraction of SSII.

Amylopectin aggregation as a function of starch phosphate content studied by size exclusion chromatography and on-line refractive index and light scattering.

Starches with a natural 65-fold span in covalently bound phosphate content were prepared from five different crops including sorghum, cassava, three potato varieties and an exotic ginger plant, Curcuma zedoaria, with extreme starch phosphate content. These starches were subjected to size exclusion chromatography with refractive index detection (SEC/RI). A simple and rapid method for starch solubilisation was used. The conditions during solubilisation (2 M NaOH) and separation (10 mM NaOH, 50 degrees C) were such as enabling > 94% recovery of the starch without detectable degradation. The aggregation properties of the starch was investigated using on line refractive index/multi angle laser light scattering (RI/MALLS) detection. Three major regions in the SEC profile were identified, consisting of large amylopectin aggregates, amylopectin particles with radius of gyration (Rg) of approx 200 nm (400 nm blocklets) and amylose. A procedure for correction of light scattering signals spread over the SEC profile as a result of aggregate tailing was developed. The significance of the relative amounts of these three molecular species on standard starch pasting parameters, as measured by a Rapid Visco Analyzer (RVA), was investigated. Starches with a high amount of amylopectin aggregates showed high peak viscosities. Moreover, very high amounts of starch bound phosphate or amylose appears to suppress the content of large aggregates resulting in low viscosity.

The distribution of covalently bound phosphate in the starch granule in relation to starch crystallinity.

Five selected starches with a 60-fold span in their content of monoesterified starch phosphate were investigated with respect to distribution of glucose 6-phosphate and glucose 3-phosphate residues, amylopectin chain length distributions and gelatinisation properties. The distribution of starch phosphate in the starch granules was determined by preparation of Nägeli dextrins followed by quantitative 31P-nuclear magnetic resonance spectroscopy. Total starch phosphate content was positively correlated to the unit chain lengths of the amylopectin as well as to the chain lengths of the corresponding Nägeli dextrins. The major part (68-92%) of the total starch phosphate content was partitioned to the hydrolysed (amorphous) parts. Starch-bound glucose 6-phosphate per milligram of starch was 2-fold enriched in the amorphous parts, whereas phosphate groups bound at the 3-position were more evenly distributed. The gelatinisation temperatures of the native starches as determined by differential scanning calorimetry were positively correlated (R(2)=0.75) to starch phosphate content, while crystallinity (gelatinisation enthalpy) and crystal heterogeneity (endotherm peak width) showed no correlations to starch phosphate content. The relations between starch molecular structure, architecture and functional properties are discussed.

Phosphorylated alpha(1-->4)Glucans as substrate for potato starch-branching enzyme I

The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using 33P-labeled phosphorylated and 3H end-labeled nonphosphorylated alpha(1-->4)glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (3H/33P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of alpha(1-->6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated alpha(1-->4)glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied 33PO43- and [U-14C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of 33P-labeled phosphorylated alpha(1-->4)glucan chains.

Alpha-glucan binding of potato-tuber starch-branching enzyme I as determined by tryptophan fluorescence quenching, affinity electrophoresis and steady-state kinetics.

The affinity of potato tuber starch-branching enzyme-I (PSBE-I) for various linear malto-oligosaccharides, cyclodextrins, (CDs) and macromolecular alpha-glucans was investigated by alpha-glucan induced fluorescence quenching of intrinsic PSBE-I tryptophan residues and by affinity electrophoresis. alpha-Glucan binding was characterised by distinct shifts towards shorter wavelengths of the PSBE-I fluorescence emission spectrum and by concomitant reductions in fluorescence intensity. The magnitudes of both the maximum shift in emission spectrum and reduction in fluorescence intensity were dependent on the alpha-glucan ligands used. Maximum Kd for a range of linear malto-oligosaccharides analysed was 0.13 mM as found at a degree of polymerisation (DP) of 13. Large differences in dissociation constants were measured for CDs with DP 6 (alpha-CD, 6.0 mM), DP 7 (beta-CD, 0.25 mM) and DP 8 (gamma-CD, 0.67 microM). The high-molecular-mass alpha-glucans amylose and amylopectin, both substrates for PSBE-I, showed apparent affinities of 0.018 and 0.066 mg/ml, respectively. Small linear and cyclic oligosaccharides competed with amylopectin in the affinity electrophoresis system and they were also competitive inhibitors for PSBE-I activity. The affinities for oligosaccharides as measured by competition were, however, about 10-fold lower than as measured by fluorescence quenching suggesting the existence of a separate oligosaccharide binding site on PSBE-I. Affinity electrophoresis revealed multiform heterogeneity in the enzyme preparation with respect to alpha-glucan interaction.

Differential expression and properties of starch branching enzyme isoforms in developing wheat endosperm.

Three forms of starch branching enzyme (BE) from developing hexaploid wheat (Triticum aestivum) endosperm have been partially purified and characterized. Immunological cross-reactivities indicate that two forms (WBE-IAD, 88 kD, and WBE-IB, 87 kD) are related to the maize BE I class and that WBE-II (88 kD) is related to maize BE II. Comparison of the N-terminal sequences from WBE-IAD and WBE-II with maize and rice BEs confirms these relationships. Evidence is presented from the analysis of nullisomic-tetrasomic wheat lines demonstrating that WBE-IB is located on chromosome 7B and that the WBE-IAD fraction contains polypeptides that are encoded on chromosomes 7A and 7D. The wheat endosperm BE classes are differentially expressed during endosperm development. WBE-II is expressed at a constant level throughout mid and late endosperm development. In contrast, WBE-IAD and WBE-IB are preferentially expressed in late endosperm development. Differences are also observed in the kinetic characteristics of the enzymes. The WBE-I isoforms have a 2- to 5-fold higher affinity for amylose than does WBE-II, and the WBE-I isoforms are activated up to 5-fold by phosphorylated intermediates and inorganic phosphate, whereas WBE-II is activated only 50%. The potential implications of this activation of BE I for starch biosynthesis are discussed.

The multiple forms of starch-branching enzyme I in Solanum tuberosum.

Western blot analysis showed the presence of three forms of starch-branching enzyme (SBE), with apparent molecular masses of 103, 97 and 80 kDa, in extracts of leaves and stored tubers of Solanum tuberosum. The 80-kDa form was absent in extracts of fresh tuber. Active 80-kDa enzyme was partially purified from stored tubers and sequence analysis showed that it, similar to the two larger enzyme forms, was an SBE-I isoform. Limited proteolysis of isolated 103-kDa SBE-I under native conditions removed approximately 200 amino acid residues from the carboxy terminus. A stable intermediate with an apparent molecular mass of approximately 80 kDa was formed. Since the 80-kDa form displayed full enzymatic activity and its circular-dichroism spectrum did not differ significantly from that of the 103-kDa enzyme, the carboxy-terminal portion of the enzyme was suggested to have an extended, unordered structure and therefore to be easily accessible to proteolysis. A cDNA sequence encoding a mature SBE-I was amplified from tuber mRNA of S. tuberosum by means of PCR. The 3' end of this sequence differed significantly from that of previously published data. PCR amplification and DNA sequencing of the 3' ends of the sbeI gene showed that four sbeI alleles exist in the cultivar studied. Two of these four alleles, sbeia and sbeIb, had slightly longer 3' ends compared with the other two, sbeIc and sbeId. The difference between the two groups of alleles was due to a partial deletion in sbeIc and sbeId of a segment duplicated in all alleles. All four alleles were expressed in leaf and tuber.

Isolation and biochemical characterization of highly purified Escherichia coli molecular chaperone Cpn60 (GroEL) by affinity chromatography and urea-induced monomerization.

Isolated Escherichia coli molecular chaperone Cpn60 (GroEL) has been further purified from tightly bound substrate polypeptides by two different procedures: (i) group-specific affinity chromatography by using the triazine dye Procion yellow HE-3G as affinity ligand, and (ii) urea-induced monomerization and subsequent chromatography. Procion yellow binds specifically to aromatic amino-acid side chains present in the majority of proteins, but has no affinity to GroEL because of its low content of aromatic residues. Some GroEL-bound polypeptides are buried within the aqueous cavity of the GroEL oligomer, whereas others are exposed on its surface and available for affinity-ligand interactions and the complex is thereby retarded on Procion yellow columns. Pure substrate-free GroEL was obtained after ion-exchange chromatography of GroEL monomers followed by reassembly of the purified monomers into functional GroEL oligomers. The final preparation contained no substrate polypeptides bound to GroEL as judged by electrophoretic analysis and lack of tryptophan fluorescence. GroEL preparations also displayed two equally strong bands on native electrophoresis suggesting the presence of two conformers. Monomers of GroEL showed heterogeneity with respect to isoelectric point and molecular mass when analysed by MALDI-MS and electrophoresis under native and denaturing conditions respectively. By use of MALDI-MS, highly accurate molecular masses of wild-type and a truncated form of GroEL were determined and verified, by comparison with their respective gene sequences.