Size Distribution of Dispersing Units in Corn Amylose

As the first step to reveal the size distribution of dispersing units in amylose, an amylose sample from corn starch was subjected to gel-filtration chromatography on columns of Sephadex G 200 and Sepharoses 6 B, 4 B and 2 B under protection of the sample from retro-gradation by the use of thiocyanate in the solvent system in the chromatography,

A considerable amount of dispersing units with unexpectedly large size was detected in the amylose sample as a. fraction which was excluded from gels of Sepharoses, though the size distribution was considered to be rather continuous covering the units with smaller size of the regular amylose in the sample. The excluded fraction involved an appreciable amount of aggregates of amylose but no usual amylopectin. Nevertheless, the fraction involved some α-1,6 glucosidic linkages susceptible to isoamylase. The digest of the fraction by the enzyme gave a product which showed size distribution similar to that of the regular size of amylose in the original sample. Both the digested product and the regular amylose had much larger size in molecular weight than the digest of amylopectin by the same enzyme.

The results indicate that the corn amylose sample is a mixture of the units with regular size amylose and those with very large one, the latter having various numbers of branches with the size of regular amylose. Thus, it is considered that the size of dispersing units in the corn amylose sample is characterized as a pattern of such continuous distribution ranging from few hundred thousand up to more than several million in terms of molecular weight.     

Conformation of amylose in water. I. Light-scattering and sedimentation-equilibrium measurements

The conformation of amylose in aqueous solution has been found to be dependent on its molecular weight. When the molecular weight of amylose is outside of the so-called “dissolving gap” described by Burchard (6500<M_r<160,000) it behaves as a random coil, whereas when its molecular weight is within the “dissolving gap,” it easily aggregates forming a rigid coil which is the B-type (retrograded) amylose. The conformation of this rigid coil is suggested to be a double helix.     

Improved methods for the structural analysis of the amylose-rich fraction from rice flour

Cooking and sensory properties of rice are largely determined by the amylose content and structure. For relationships between functional and structural properties, a more accurate method to determine the structure of amylose is required. Here we calibrate size exclusion chromatography (SEC) columns, using Mark-Houwink parameters for linear starch and pullulan standards, to obtain the true molecular weight distribution of linear starch. When the molecular weight distribution is reported relative to pullulan, rather than the actual molecular weight which is readily obtained from universal calibration, it is seen that the molecular weights of longer amylose chains are greatly underestimated. We validate the SEC method to enable the measurement of the hydrodynamic volume distribution of the starch by examining reproducibility and recovery. Analysis of the starch in the sample pre- and post-SEC shows that 20% of the carbohydrate is not recovered. Comparison of the weight-average degree of polymerization, X(w), of (undebranched) starch of pre- and post-SEC is made using iodine binding as well as Berry plots of data from multi-angle laser light scattering (MALLS). These both show that current SEC techniques for starch analysis lead to significant loss of high molecular weight material. Indeed, for the systems studied here, the values for X(w) after SEC are about three times lower than those before SEC. Iodine-starch complexes of pre- and post-SEC samples reveals that the SEC techniques give reliable data for the amylose fraction but not for amylopectin. We address reports in the literature suggesting that the conventional isoamylase method for debranching starch would lead to incomplete debranching and thus incorrect molecular weight distributions. However, it is shown using (1)H NMR that isoamylase can completely debranch the amylose (to within the detection limit of 0.5%), and by SEC that successive incubation with isoamylase, alpha-amylase, and beta-amylase can degrade the amylose-rich fraction completely to maltose. We develop a method to obtain a hot water soluble fraction (HWSF), rich in undamaged amylose molecules, directly from rice flour, avoiding the structural degradation of previous techniques. With appropriate sample handling, the formation of associations between starch chains is minimized. With the combination of calibrated and validated SEC methods, and an improved extraction of amylose from rice, the X(w) for both HWSF and debranched HWSF are found to be much larger than has previously been reported.     

The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy

The molecular deposition of starch extracted from normal plants and transgenically modified potato lines was investigated using a combination of light microscopy, environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). ESEM permitted the detailed (10 nm) topographical analysis of starch granules in their hydrated state. CLSM could reveal internal molar deposition patterns of starch molecules. This was achieved by equimolar labelling of each starch molecule using the aminofluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Starch extracted from tubers with low amylose contents (suppressed granule bound starch synthase, GBSS) showed very little APTS fluorescence and starch granules with low molecular weight amylopectin and/or high amylose contents showed high fluorescence. Growth ring structures were sharper in granules with normal or high amylose contents. High amylose granules showed a relatively even distribution in fluorescence while normal and low amylose granules had an intense fluorescence in the hilum indicating a high concentration of amylose in the centre of the granule. Antisense of the starch phosphorylating enzyme (GWD) resulted in low molecular weight amylopectin and small fissures in the granules. Starch granules with suppressed starch branching enzyme (SBE) had severe cracks and rough surfaces. Relationships between starch molecular structure, nano-scale crystalline arrangements and topographical-morphological features were estimated and discussed.     

Imaging of carrageenan macrocycles and amylose using noncontact atomic force microscopy

Samples of kappa-carrageenan, iota-carrageenan, and synthetic amylose have been examined by atomic force microscopy (AFM). All samples were spray deposited from aqueous solutions onto freshly cleaved mica, air dried, and imaged in air using noncontact atomic force microscopy (NCAFM). Images of single stranded amylose and carrageenan are presented. At relatively low polymer concentrations in the presence of NaCl iota-carrageenan formed circles that appear to be predominantly head-to-tail associated unimeric duplex (double stranded) structures. At higher iota-carrageenan concentrations the polymer forms circles and aggregates that appear to involve dimeric duplex structure. Direct comparison of synthetic amylose molecular weights determined from NCAFM images with results from solution measurements showed that NCAFM provides an excellent way to measure amylose molecular weight and molecular weight distribution. It is shown that synthetic amylose is single stranded in aqueous solution and that the chain length distribution is broader than the Poisson distribution anticipated from polymerization theory.     

Retention of components in a mixture of volatile organic substances by maltodextrins

The effect of composition and origin of maltodextrins on the retention of 38 components in a mixture of volatile organic substances (odorants) during 6-month storage was studied by means of capillary gas-liquid chromatography. The retention of esters increased with an increase in their molecular weight. The retention of lactones, phenols, linalool, menthone, and damascone was 75-85%. Storage of aldehydes was accompanied by oxidation, and the retention of these substances did not exceed 55%. The retention of odorants increased with a decrease in the molecular weight of maltodextrins. The maximum retention was typical of maltodextrin from amylopectin starch not containing amylose.     

Molecular weight determination of peptides by high-performance gel permeation chromatography

A method for molecular weight determination of small peptides using Bio-Sil TSK 20 and Bio-Gel TSK 125 columns is described. The TSK 20 column provided a good separation of the standard peptides in the range from 1000-10,000 with an accuracy of less than 5% from the calculated regression line. Two combined TSK 125 columns allowed a reliable molecular weight determination in the range from 800 to 3500.     

Generation of corticosteroid binder IB from binder II by a sulfhydryl dependent renal cytosolic factor

It has long been debated whether binder IB represents a unique form of the glucocorticoid receptor or is derived from the larger molecular weight form, binder II, by limited proteolysis. Transformed glucocorticoid receptors in kidney, liver and mixed kidney/liver cytosols were examined using anion exchange and gel filtration chromatography. The transformed receptor in liver cytosols chromatographs as binder II on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of approx 6.0 nm. The transformed receptor in kidney cytosols chromatographs as binder IB on DEAE-Sephadex A-50 anion exchange columns and has a Stokes radius of 3.0-4.0 nm (3.2 nm on agarose; 3.0-4.0 nm on Sephadex G-100). Using cytosols prepared from mixed homogenates (2 g kidney plus 8 g liver tissue), our experiments show that binder II is converted to a lower molecular weight form (Rs = 3.2 nm on agarose; Rx = 3.9 nm on Sephadex G-100) that is identical to binder IB in its elution position from DEAE-Sephadex anion exchange resin. Identical results are obtained using kidney/liver/cytosols mixed in vitro in which only the hepatic receptor, binder II, is labelled with [3H]TA. These results support the hypothesis that the renal receptor, binder IB, is a proteolytic fragment of binder II and does not represent a polymorphic form of the glucocorticoid receptor. The renal converting activity is dependent on free-SH for full activity but is insensitive to the protease inhibitors leupeptin, antipain, and PMSF. The conversion of hepatic binder II to binder IB in in vitro mixing experiments can be prevented if kidney cytosol is gel filtered on Sephadex G-25 and the eluted macromolecular fraction is adjusted to 10 mM EGTA (or EDTA) prior to mixing with the [3H]TA labelled hepatic cytosol.     

Temperature effect on retrogradation rate and crystalline structure of amylose

Commercial potato amylose was used to study temperature effects on the retrogradation of amylose solutions (3.5mg/ml). The retrogradation rate decreased as incubation temperature increased (5 to 45 °C). The degree of retrogradation within 24 h decreased from 58.8 to 7.1% as incubation temperature increased from 5 to 45 °C. In the amylose solution, different-sized molecular subfractions retrograded at different rates. After incubating at 5 °C for 100 days, the majority of the amylose molecules retrograded and precipitated from the solution; at 45 °C, only amylose of the small-molecular subfraction (number average, DP_n = 110; weight average, DP_w = 150) retrograded and precipitated. Entanglement of molecules was observed in size exclusion chromatograms. The morphology of retrograded amylose observed by using a scanning electron microscope differed with the retrogradation temperature. The chain length of amylose crystalline segments, prepared by hydrolysis of retrograded amylose, showed a narrow distribution (polydispersity from 1.21 to 1.67). The chain lengths of resistant segments increased DP_n from 39 to 52 and DP_w from 47 to 72 for α-amylolysis and DP_n from 34 to 40 and DP_w from 48 to 67 for 16% sulfuric acid hydrolysis, when incubation temperature increased from 5 to 45 °C.     

Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice

A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a).     

Biological fate of a polydisperse acrylate polymer in anaerobic sand-medium transport

Soluble polyacrylate (PA), a polydisperse mixture of polyacrylate polymers, is strongly adsorbed and biodegradable. Biotic fate studies were carried out with once-through columns containing sand colonized with anaerobic biomass previously grown in a methanogenic fluidized bed. A fraction of soluble PA having a weight-average molecular weight of 16,700 and a range of molecular weight from 10³ to 10⁵ was biologically removed and mineralized to CO₂. Due to its polydisperse nature, the breakthrough curve had a gradual increase to an apparent steady-state removal of approximately 60% near one day when the liquid detention time was 21 minutes. Modeling successfully explained the observed breakthrough result when the fraction was divided into components having a wide range of retardation factors (R): about 25% was strongly adsorbed (R=200 and 500), 45% was moderately adsorbed (R=50 and 100), and 30% was weakly adsorbed (R=1–10). In this study, in which active biomass already was present from utilization of a primary substrate (glucose here), equilibrium adsorption increased the time to breakthrough, which also reduced the exiting concentration by increasing the substrate contact time.     

Similar molecular properties of granulocyte-macrophage colony-stimulating factors produced by different mouse organs in vitro and in vivo.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was partially purified from post-endotoxin serum and conditioned media produced by organs from both normal and endotoxin-injected C57BL mice. The organs used to condition medium were heart, thigh muscle, salivary gland, thymus, spleen, kidney, brain, and femur shaft. The charge properties, molecular weights, and concanavalin A binding profiles of these GM-CSFs were analyzed and compared to purified mouse lung GM-CSF. All the GM-CSFs examined were shown to be gycoproteins since a proportion of the activity (80 to 100%) bound to concanavalin A-Sepharose. The organ-conditioned medium GM-CSFs were purified (3- to 13-fold) by absorption to calcium phosphate gel and chromatography on DEAE-Sepharose (further 2- to 10-fold). Analysis of the DEAE-Sepharose elution profiles indicated that there were two major charge species of GM-CSF eluting at conductivities of 10 and 14 mmho. These partially purified GM-CSFs showed considerable differences in their apparent molecular weights on Sephacryl S-200 (37,000 to 200,000). However, these differences could be eliminated by treating the GM-CSFs with neuraminidase and performing molecular sizing experiments under dissociating conditions (Sepharose CL-6B, 6 M guanidine hydrochloride). Although some of the GM-CSFs showed anomalously high molecular weights (40,000) on gel filtration columns, even under dissociating conditions, this appeared to be due to properties of the sialic acid residues. After neuraminidase treatment all of the conditioned medium GM-CSFs eluted from DEAE-Sepharose as a single peak of biological activity at a conductivity of 10 mmho and from gel filtration columns in the presence of 6 M guanidine hydrochloride as a single molecular weight species of approximately 23,000. GM-CSF from post-endotoxin serum (produced in vivo) eluted from the gel filtration column with an apparent molecular weight of 39,000, but analysis using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that this GM-CSF also had an apparent molecular weight of 23,000.     

Structure and proteolysis of the growth hormone receptor on rat hepatocytes

125I-Labeled human growth hormone is isolated in high molecular weight (Mr) (300,000, 220,000, and 130,000) and low molecular weight complexes on rat hepatocytes after affinity labeling. The time-dependent formation of low molecular weight complexes occurred at the expense of the higher molecular weight species and was inhibited by low temperature or inhibitors of serine proteinases. Exposure to reducing conditions induced loss of Mr 300,000 and 220,000 species and augmented the amount of Mr 130,000 complexes. The molecular weight of growth hormone (22,000) suggests that binding had occurred with species of Mr 280,000, 200,000, and 100,000. Two-dimensional gel electrophoresis demonstrated that the 100,000-dalton receptor subunit is contained in both the 280,000- and 200,000-dalton species. Reduction of interchain disulfide bonds in the growth hormone receptor did not alter its elution from gel filtration columns, but intact, high molecular weight receptor constituents were separated from lower molecular weight degradation products. Digestion of affinity-labeled growth hormone-receptor complexes with neuraminidase increased the mobility of receptor constituents on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These observations show that the growth hormone receptor is degraded by hepatic serine proteinases to low molecular weight degradation products which can be separated from intact receptor by gel filtration. Intact hormone-receptor complexes are aggregates of 100,000-dalton sialoglycoprotein subunits held together by interchain disulfide bonds and by noncovalent forces.     

Enantiomeric resolution by HPLC of axial chiral amides using amylose tris[(S)-1-phenylethylcarbamate]

The enantiomeric resolution of a series of N-arylamides was examined on amylose tris[(S)-1-phenylethylcarbamate] coated onto aminopropylated 7 μm silica with 500 Å diameter pores and on naked silica 5 μm particle size with 500 Å diameter pores. The enantiomeric resolution obtained for this series was excellent on both columns. The enantioselectivity of cellulose and amylose tris (3,5-dimethylphenylcarbamate) coated onto APS-Hypersil (120 Å pore size, 5 μm particle size) was also investigated for this series of compounds. Chirality 9:109–112, 1997. © 1997 Wiley-Liss, Inc.     

Super-absorbent polymers from starch-polyacrylonitrile graft copolymers by acid hydrolysis before saponification

Graft copolymers (SPAN) of polyacrylonitrile (PAN) onto starch were prepared from gelatinized starch varieties with ammonium ceric nitrate as an initiator. The molecular weight of the PAN branches increased for the varieties of starches in the order high amylose maize starch < maize starch < waxy maize starch. SPAN samples were saponified with aqueous NaOH, and the aqueous solution of the resulting polymer (HSPAN) was cast into film in a forced-air oven at 35°C. The water absorbency of the HSPAN film formed from waxy maize starch was the highest (1200 g H₂O (g dry sample)⁻¹) and that from high amylose maize starch was the lowest (530 g g⁻¹). SPAN samples from maize starch were partially hydrolyzed with dilute hydrochloric acid. The resulting polyacrylonitriles with low molecular weight starch end groups (LSPAN) were also saponified. The resulting saponified product (HLSPAN) was cast into film. The absorbencies of HLSPAN films were found to be far larger (up to 6000 g g⁻¹) than those of the corresponding HSPAN films. The absorbency increased with increasing molecular weight of PAN in the initial SPAN up to a molecular weight of 1−1·5 × 10⁶. The absorbency decreased significantly when HSPAN and HLSPAN films were subjected to heat treatment at 135°C or above. The crosslinks present in HSPAN and HLSPAN films prepared at 35°C and those formed during heat treatment were considered to have different structures: the former formed between carbohydrate alkoxide ions and nitrile groups at the early stages of saponification and the latter formed between carbohydrate and copoly(acrylate-acrylamide) chains and/or between copoly(acrylate-acrylamide) chains.     

Protein S1 from Escherichia coli ribosomes: an improved isolation procedure and shape determination by small-angle X-ray scattering.

Ribosomal protein S1 from Escherichia coli was studied in solution by small-angle X-ray scattering and the following parameters were obtained. The radius of gyration R = 8.0 +/- 0.2 nm; largest diameter D = 28 nm; molecular weight = (8--9) x 10(4). The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross-section Rq1 = 2.5 +/- 0.1 nm and Rq2 = 1.05 +/- 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X-ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41--54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape. Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X-ray irradiation.     

Molecular weight characterization of starch and modified starches as tricarbanilate derivatives

Industrial corn starches modified with an oxidant, acid or enzyme have been analyzed by a high-performance size-exclusion chromatography (HPSEC) technique. The procedure involves derivatization of the starch to the corresponding tricarbanilate with separation afforded by commercially available polystyrene-divinylbenzene (PS-DVB) gel columns. Separation time was under 30 min and UV detection at 235 nm allows the analysis of microgram quantities. The elution behavior of amylose and amylopectin tricarbanilates appears to depend on allowed conformational states. Amylopectin tricarbanilate (APTC) is hindered by branching and elutes much earlier than the exclusion limit of the column system according to linear polystyrene standards. Observation of an apparent hydrolysis resistant region of starch and the effects of the various hydrolytic treatments are discussed. Relative molecular weight data are presented utilizing the carbanilate of monodisperse pullulan polysaccharides as primary standards.     

Purification of cartilage-derived growth factor by heparin affinity chromatography

Cartilage-derived growth factor (CDGF) was found to bind tightly to columns of immobilized heparin and could be eluted with concentrations of salt in the order of 1.6-1.8 M NaCl. The molecular weight of CDGF was estimated to be 18,000-20,000 by high performance liquid-size exclusion chromatography. The affinity of CDGF for heparin greatly facilitated its purification. Highly purified CDGF active at about 1-2 ng/ml was obtained when crude cartilage extract was applied to heparin-Sepharose and the growth factor activity was recycled over heparin-Sepharose two more times. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver stain visualization of highly purified CDGF showed one major polypeptide band with a molecular weight of about 19,000 containing over 95% of the protein and one minor polypeptide band containing the rest of the protein. Only the Mr 19,000 polypeptide was active after elution from the polyacrylamide gel. Although CDGF bound tightly to immobilized heparin, it did not bind to immobilized chondroitin sulfate or hyaluronic acid. In addition, CDGF bound to heparin much more tightly than did platelet-derived growth factor even though these two growth factors had similar isoelectric points of about 10. These results suggest that the binding of CDGF to heparin was due to a specific affinity of the 2 molecules for each other.     

Biosynthesis of sulphated macromolecules by rabbit lens epithelium. I. Identification of the major macromolecules synthesized by lens epithelial cells in vitro

Rabbit lens epithelial cells synthesize and secrete a variety of [35S]sulphate-labeled glycoconjugates in vitro. Associated with the cell layer, and with the medium, was a high molecular weight glycoconjugate(s) that contained heparan sulphate which was apparently covalently linked to sulphated glycoprotein. This component(s) was eluted in the void volume of a Sepharose CL-2B column and could not be fractionated by detergent treatment or extraction with lipid solvents. The cell layer also contained glycosaminoglycans (72% heparan sulphate, 28% chondroitin sulphate), as well as a small proportion of a low molecular weight sulphated glycoprotein. The major 35S-labeled species secreted into the medium were sulphated glycoproteins with approximate molecular weights of 120,000 and 35,000 together with a heparan sulphate proteoglycan. This proteoglycan could be precipitated from the culture medium with 30% saturated (NH4)2SO4 and eluted from Sepharose CL-4B columns at approximately the same position (Kav = 0.15) as heparan sulphate proteoglycans described in the basement membrane of the EHS "sarcoma" (Hassell, J. R., P. G. Robey, H. J. Barrach, J. Wilczek, S. I. Rennard, and G. R. Martin, 1980, Proc. Natl. Acad. Sci. USA, 77:4494-4498) and of the mouse mammary epithelium (David, G., and M. Bernfield, 1981, J. Cell Biol., 91:281-286). Its presence in the culture medium was unanticipated but may be explained by the inability of these cultures to deposit a basement membrane when grown on a plastic surface. The relationship of this heparan sulphate proteoglycan to the lens epithelial basement membrane is the subject of the following paper.     

Determination of depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using a fluorimetric 2-p-toluidinylnaphthalene-6-sulfonate/flow-injection analysis system

This study reports on the determination of the depolymerization kinetics of amylose, amylopectin, and soluble starch by Aspergillus oryzae alpha-amylase using flow-injection analysis with fluorescence detection and 2-p-toluidinylnaphthalene-6-sulfonate as the fluorescent probe. The experimental data points, corresponding to the evolution of the concentration of "detectable" substrate with depolymerization time, were fit to a single exponential decay curve in the case of amylose and to a double exponential decay curve in the cases of amylopectin and soluble starch. For all the assayed substrates, the determined depolymerization rates at time zero correlated well with the initial enzyme and substrate concentrations through the usual Michaelis-Menten hyperbola. Therefore, this methodology allows the determination of alpha-amylase activity using any of these substrates. For amylopectin and soluble starch, the value of the total depolymerization rate at any depolymerization time was the result of the additive contribution of two partial depolymerization rates. In contrast, the total depolymerization rate for amylose was always a single value. These results, in conjunction with the relative time evolution of the two partial depolymerization rates (for amylopectin and soluble starch), are in good agreement with a linear molecular structure for amylose, a "grape-like" cluster molecular structure for amylopectin, and an extensively degraded grape-like cluster structure for soluble starch.