Maltooligosaccharides Research Articles

Starch Digestion Products Activate Enteroendocrine L-cells and Their Ileal Delivery Through the Diet Contributes to Weight Management in Mice (P08-005-19)

ObjectivesOur laboratory has recently shown that slowly digestible starch (SDS) that locationally digests to the ileum activates the gut-brain axis and reduces food intake in obese animals. Glucagon-like peptide-1 (GLP-1), oxyntomodulin (OXM), and peptide YY (PYY) are the main appetite-suppressing (anorexigenic) peptides of the intestinal enterendocrine L-cells that regulate postprandial insulin levels and satiety signals. We investigated the in vitro L-cell chemosensation of α-amylase starch digestion products, named maltooligosaccharides (MOS), and using SDS to deliver MOS in vivo. MethodsMouse (STC-1) and human (NCI-H716) cells were used to test chemosensation response and release of GLP-1, OXM and PYY after MOS treatment. Differential gene expression and comparable global protein profiling of STC-1 cell treated with MOS was tested using RNA sequencing and LC-MS/MS analysis. Alginate-entrapped SDS microspheres that digest distally into the ileum were used to examine the role of SDS in the intervention and prevention of obesity in C57BL/6 J obese and lean mice, respectively. Body weight, food intake and body composition were monitored periodically. ResultsMOS exhibited significantly higher stimulatory effect on GLP-1 and OXM secretion in mouse and human L-cells, respectively, compared to glucose. Multi-omics analysis showed that MOS induced exocytosis of GLP-1- and OXM-containing vesicles and did not induce positive regulation of the proglucagon gene suggesting that secretion, but not synthesis, of the proglucagon gene products was enhanced by MOS. In vivo Results showed that 20% SDS in low-fat diets significantly improved weight loss and food intake reduction in obese mice. Similarly, 15% SDS in high-fat diets showed significant reduction in body fat %, increase in lean body mass, and considerable reduction in weight gain rate and food intake in lean mice fed on high-fat diets. ConclusionsWe propose several insights into L-cell sensation of dietary starch-degraded MOS delivered by the consumption of slowly digestible starch. MOS exhibit unique influences on L-cell sensitivity and gut hormone productivity. The intricate role of dietary carbohydrates on gut physiological response, related to satiety and food intake could be a new approach for design of foods for obesity prevention. Funding SourcesWhistler Center for Carbohydrate Research, Purdue University. Supporting Tables, Images and/or Graphs▪▪

Regional Differences in Taste Responsiveness: Effect of Stimulus and Tasting Mode

Previous studies have shown that there are differences in taste responses between various regions of the tongue. Most of those studies used a controlled "passive" tasting mode due to the nature of investigation. However, food is rarely tasted in a passive manner. In addition, recent studies have suggested that humans can taste maltooligosaccharides (MOS) and that the gustatory detection of MOS is independent of the known sweet receptor. It is unknown whether regional differences in responsiveness to MOS exist. This study was set up to revisit previous work by investigating the effects of tasting mode ("passive" vs. "active") on regional differences in taste responsiveness to sucrose, monopotassium glutamate (MPG), and quinine, while also investigating potential regional differences in responsiveness to MOS. The stimuli were applied to 1 of 4 target areas, the left and right sides of the front and back of the tongue, using cotton-tipped swabs. In the passive tasting condition, the front of the tongue was found to be more responsive to both sucrose and MOS, but no regional differences were seen for quinine and MPG. In contrast, in the active tasting condition, the back of the tongue was found to be more responsive to quinine and MPG, but no differences were found for sucrose or MOS. These findings indicate that there are regional differences in taste responsiveness between the front and back of the tongue and that regional responsiveness is dependent on stimulus and tasting mode.

Bioconversion of sucrose to maltooligosaccharides by the synergistic action of amylosucrase and α-amylase

Abstract Maltooligosaccharides (MOS), oligosaccharides composed of 2 to 10 glucopyranose subunits linked by α-1,4 bonds, are promising ingredients possessing low sweetness, low osmolality, high water-holding capacity, and crystallization-prevention ability. For improved the cost effectiveness, a novel process for MOS preparation was developed by utilizing sucrose as a raw material instead of starch. In this work, a novel α-amylase from Pseudomonas mendocina (PM-Amy) was characterized and applied to produce the MOS from α-1,4-glucans obtained by the amylosucrase treatment of sucrose. PM-Amy showed a maximum activity at a pH of 6.0 and 55 °C. In the one-pot enzymatic reaction, a maximum MOS yield of 22.8% (w/w) was achieved, while the highest MOS yield (34.1%, w/w) was achieved from the two-step dual enzyme reaction.

Preparation and characterization of isolated low degree of polymerization food-grade maltooligosaccharides

Research involving human responses to the consumption of starch and its hydrolysis products would benefit from convenient sources of well defined, low cost, food-grade maltooligosaccharides (MOS). This report addresses such need by presenting an approach to obtain aforementioned MOS. A chromatography-ready MOS sample containing proportionately high amounts of low degree of polymerization (DP) MOS is initially prepared from commercially-available maltodextrins (MD) by taking advantage of the DP-dependent differential solubility of MOS in aqueous-ethanol solutions. The low DP-enriched MOS preparation is subsequently fractionated via preparative column chromatography using cellulose-based stationary phases and step-gradient aqueous-ethanol mobile phases. The resulting fractions yielded isolated food-grade MOS ranging in DP from 3 to 7. NMR spectra of isolated MOS indicated minimal amounts of branched saccharides. Typical yields from a single fractionation protocol (2 g MD starting material), including solvent partitioning through preparative chromatography, ranged from ∼40 mg for DP 4, 5, and 7 to ∼100 mg for DP 3 and 6.

Plastidial α-glucan phosphorylase 1 complexes with disproportionating enzyme 1 in Ipomoea batatas storage roots for elevating malto-oligosaccharide metabolism.

It has been proposed that malto-oligosaccharides (MOSs) are possibly recycled back into amylopectin biosynthesis via the sequential reactions catalyzed by plastidial α-glucan phosphorylase 1 (Pho1) and disproportionating enzyme 1 (Dpe1). In the present study, the reciprocal co-immunoprecipitation experiments using specific antibodies against Pho1 and Dpe1 demonstrated that these two enzymes can form a complex (the PD complex) in Ipomoea batatas storage roots. The immunohistochemistry analyses also revealed the co-localization of Pho1 and Dpe1 in the amyloplasts, and the protein levels of Pho1 and Dpe1 increased gradually throughout sweet potato storage root development. A high molecular weight PD complex was co-purified from sweet potato storage root lysates by size exclusion chromatography. Enzyme kinetic analyses showed that the PD complex can catalyze maltotriose and maltotetraose to generate glucose-1-phosphate in the presence of inorganic phosphate, and it also performs greater Dpe1 activity toward MOSs than does free form Dpe1. These data suggest that Pho1 and Dpe1 may form a metabolon complex, which provides elevated metabolic fluxes for MOS metabolism via a direct transfer of sugar intermediates, resulting in recycling of glucosyl units back into amylopectin biosynthesis more efficiently.

Compressed food with added functional oligopeptides improves performance during military endurance training.

Oligosaccharide or oligopeptide supplementation may have a significant impact on endurance performance. This study evaluated the effects of adding maltooligosaccharides (MO) or soy oligopeptides (SO) to compressed food (CF) on the physical response of soldiers to daily military training. Twelve soldiers were randomized to four diet groups: regular meals, CF, CFMO, and CFSO (crossover design). They participated in exercise tests including 90 minutes running at 55-65% VO2max and exhaustive running. Heart rates, rating of perceived exertion (RPE), and blood and urine samples were collected during exercise and recovery. The recovery heart rates were significantly lower with the CFMO diet compared with the other diets. Compared with all other diets, blood glucose levels were higher, post-exercise blood lactate levels were lower, and lactate clearance during recovery was higher with the CFMO diet, followed by the CFSO diet. Post-exercise levels of erythrocytes and hematocrit were significantly higher with the CFSO diet. Post-exercise urine specific gravity was lower with the CFMO diet and urine pH was decreased with the CFSO diet. Blood urea nitrogen (BUN) and uric acid (UA) were significantly higher with the CFSO diet than with the other diets. There was no significant difference in skeletal and cardiac muscle injury indices and RPE among diets. CFMO led to better heart rate recovery, improved and maintained blood glucose and increased removal of blood lactate. CFSO accelerated removal of blood lactate during recovery, maintained oxygen supply, and increased fluid retention.

Evaluation of oligosaccharide profiles in selected cooked tubers and roots subjected to in vitro digestion

The influence of the preparation method and the effect of in vitro digestion on the fructo-oligosaccharides (FOS) and malto-oligosaccharides (MALTOS) of sweet potato (Ipomoea batatas Lam), mandioquinha (Arracacia xanthorrhiza), white yam (Dioscorea alata L.), cassava (Manihot esculenta Crantz), and taro (Colocasia esculenta) were evaluated. Tubers/roots were cooked in two ways: whole/unpeeled and peeled/in cubes. In vitro digestion was performed on whole/unpeeled cooked vegetables. An oligosaccharide analysis was carried out by HPAE-PAD. The largest amounts of oligosaccharides were found in the whole/unpeeled cooked tubers/roots. Sweet potato showed the highest content of FOS (29.08 ± 1.60 mg/100 g) and the highest concentrations of MALTOS were observed in white yam (113.70 ± 3.41 mg/100 g). In vitro digestion promoted an increase in maltotriose in all tubers/roots. Sweet potato FOS were not changed by digestion. However, in mandioquinha, white yam, and cassava, FOS levels were higher after digestion. The increase in MALTOS after in vitro digestion may be associated with both the hydrolysis of oligosaccharides with a higher degree of polymerization and starch. FOS contents increased after digestion possibly by hydrolysis of inulin or release from the food matrix. Sweet potato, cassava, and white yams can contribute to an increased intake of prebiotics.

Enzymatic synthesis using immobilized Enterococcus faecalis Esawy dextransucrase and some applied studies

Dextrans enzymatic synthesis by immobilized Enterococcus faecalis Esawy dextransucrase was studied. Different parameters, such as: enzyme protein concentration (EPC), substrate concentration (SC), temperature and reaction time were evaluated. EPC played a fundamental role in controlling dextran molecular size with 0.1% dextran in reaction mixture. Dextran 38,397 and 125,471Da were yielded at EPC 4.78 and 5.78mg, respectively. Proper dextrans (73,378 and 117,521Da) demanded in pharmaceutical applications were achieved at 6% and 12% sucrose concentrations and at 4.78 and 5.78mg EPC, respectively. Optimum temperature for conversion of glucose to dextran was 30°C (73% and 80% at 5.78 and 4.78mg EPC, respectively). Varieties of maltooligosaccharides (MOS) were yielded by synergistic cooperation between sucrose and maltose. Six MOS and three dextrans samples in vitro have prebiotic effect on Lactobacillus casei with degree of variation. Two samples of MOS with different degree of polymerization (DP) and three samples of dextran with different molecular weight (MW) reported different fibrinolytic activity.

On the use of differential solubility in aqueous ethanol solutions to narrow the DP range of food-grade starch hydrolysis products

Considerable research is focused on understanding the functionality of starch hydrolysis products (SHP) consisting of glucose, maltose, maltooligosaccharides (MOS), and maltopolysaccharides (MPS). A confounding factor in this research is the high molecular dispersity of commercially available SHP. The study presented herein characterizes a flexible fractionation approach for lowering the dispersity of such products. This was accomplished by fractionating a corn syrup solids (CSS) preparation based on the differential solubility of its component saccharides in aqueous-ethanol solutions. Products obtained from selected fractionations were characterized with respect to degree of polymerization (DP; liquid chromatography), dextrose equivalency (reducing sugar assays), and prevalence of branching (NMR). Glucose and maltose were preferentially removed from CSS using high (⩾90%) ethanol extractants. Preparations with relatively narrow ranges of MOS, lower DP MPS, and higher DP MPS were obtained through repetitive 70%-ethanol extractions. Linear, as opposed to branched, MOS and MPS were preferentially extracted under all conditions tested.

Production optimization, purification and characterization of α-amylase from thermophilicBacillus licheniformis TSI-14

Bacillus licheniformis TSI-14 was isolated form a natural hot water reservoir of TulsiShyam in the Gir Forest, Gujarat (India). The bacterium was identified by 16S rDNA gene sequencing. Amylase production was optimized using an RSM approach (Response Surface Methodology; variables: temperature, pH, medium yeast extract and peptone, and incubation period). These data were refined using Central Composite Design (CCD). The conditions deduced for the maximal α-amylase production were 50°C, pH 7.0, and incubation times of 2 days. A 4.6 fold increase in α-amylase was achieved. The molecular weight of the purified enzyme is 31 KDa by SDS-PAGE. It is active at elevated temperatures such as 90°C, and it prefers pH values around neutral. Optimum catalysis and stability occurred at 70°C and pH 7. There is a strong Mg++ dependency evident; other metal ions show inhibition to a lesser or greater extent. The chelator EDTA completely inhibits while inhibitors urea and beta-mercaptoethanol show inhibition to a greater extent; thiourea enhances activity. Malto–oligosaccharides are intermediate products along with maltose as expected of a α-amylase.

Gluco-oligomers initially formed by the reuteransucrase enzyme of Lactobacillus reuteri 121 incubated with sucrose and malto-oligosaccharides

The probiotic bacterium Lactobacillus reuteri 121 produces a complex, branched (1 → 4, 1 → 6)-α-D-glucan as extracellular polysaccharide (reuteran) from sucrose (Suc), using a single glucansucrase/glucosyltransferase (GTFA) enzyme (reuteransucrase). To gain insight into the reaction/product specificity of the GTFA enzyme and the mechanism of reuteran formation, incubations with Suc and/or a series of malto-oligosaccharides (MOSs) (degree of polymerization (DP2-DP6)) were followed in time. The structures of the initially formed products, isolated via high-performance anion-exchange chromatography, were analyzed by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry and 1D/2D (1)H/(13)C NMR spectroscopy. Incubations with Suc only, acting as both donor and acceptor, resulted in elongation of Suc with glucose (Glc) units via alternating (α1 → 4) and (α1 → 6) linkages, yielding linear gluco-oligosaccharides up to at least DP ~ 12. Simultaneously with the ensemble of oligosaccharides, polymeric material was formed early on, suggesting that alternan fragments longer than DP ~ 12 have higher affinity with the GTFA enzyme and are quickly extended, yielding high-molecular-mass branched reuteran (4 × 10(7) Da). MOSs (DP2-DP6) in the absence of Suc turned out to be poor substrates. Incubations of GTFA with Suc plus MOSs as substrates resulted in preferential elongation of MOSs (acceptors) with Glc units from Suc (donor). This apparently reflects the higher affinity of GTFA for MOSs compared with Suc. In accordance with the GTFA specificity, most prominent products were oligosaccharides with an (α1 → 4)/(α1 → 6) alternating structure.

Accumulation and degradation of starch in carrot roots

Carrot (Daucus carota L.) roots are traditionally considered as containing negligible amounts of starch. Depending on the type of extraction medium used to remove interfering sugars from root tissue however, the yield of starch content varies. To re-examine the role of starch in carrots, high-starch cultivars were identified and their starch and sugar content monitored during root development in the field and during storage. Starch up to 150 or even 180mgg−1 dry weight accumulated in some of these cultivars. Low-temperature storage (2°C) decreased the starch content dramatically and concomitantly increased the sucrose content. Sensory triangle tests and ranking tests of sweetness performed with juices produced from carrot roots revealed significant differences between freshly harvested and stored roots, indicating a gain in sweetness achieved by starch mobilization. Post-harvest sweetening of carrots was accompanied by a transient increase in total amylase activity and malto-oligosaccharides (MOS) content. In contrast, if roots were stored at room temperature (19°C), there was also a decrease in starch content and an increase in sucrose content, but total amylase activity and MOS content declined, suggesting different metabolic pathways at the two temperatures. The possible role of starch in sugar accumulation and in stress adaptation of carrot is discussed. It is concluded that starch is a key reserve carbohydrate in carrot.

Identification of Fructo- and Malto-oligosaccharides in Cured Tobacco Leaves (Nicotiana tabacum)

Fructo-oligosaccharides (FOSs) and malto-oligosaccharides (MOSs) in cured tobacco leaves ( Nicotiana tabacum ) were detected and quantified using high-performance liquid chromatography (HPLC) coupled with electrospray ionization (ESI) tandem mass spectrometry (MS/MS). These oligosaccharides were present in several tobacco materials including flue-cured tobacco, sun/air-cured tobacco, and cut filler of commercially available tobacco products, but were not detected in air-cured tobacco. The changes in these oligosaccharides during storage were also investigated. The results revealed that MOSs simply decreased under a warm atmosphere and FOSs increased. In addition, the formation of FOSs in cured tobacco leaves occurred in the presence of sucrose. With regard to FOSs, it has been reported that green tobacco leaves do not contain FOSs such as kestose, nystose, and fructosyl-nystose. The results of a model test suggested that the changes in FOS amount were caused by enzymatic reactions.

Structural characterization of linear isomalto-/malto-oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase enzyme from Lactobacillus reuteri 121

Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1→4) also (α1→6) glycosidic linkages, and it was classified as a 4,6-α-glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of MOSs and their corresponding alditols [degree of polymerization, DP2(-ol)-DP7(-ol)] were carried out, and (purified) products were structurally analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and one-/two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto-/malto-oligomers, in the case of DP7 up to DP >35.

Expression and characterization of an extremely thermostable β-glycosidase (mannosidase) from the hyperthermophilic archaeon Pyrococcus furiosus DSM3638

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF0356) similar to the enzymes in glycoside hydrolase family 1. This β-glycosidase, designated PFTG (P. furiosus thermostable glycosidase), was cloned and expressed in Escherichia coli. The expressed enzyme was purified by heat treatment and Ni-NTA affinity chromatography. The gene was composed of 1,452 bp encoding 483 amino acids for a protein with a predicted molecular mass of 56,326 Da. The temperature and pH optima were 100°C and 5.0 in sodium citrate buffer, respectively. The substrate specificity of PFTG suggests that it possesses characteristics of both β-galactosidase and β-mannosidase activities. However, through kinetic studies by ITC (Isothermal Titration Colorimetry) which is very sensitive method for enzyme kinetics, PF0356 enzyme revealed the highest catalytic efficiency toward p-nitrophenyl-β-d-mannopyranoside (3.02 k(cat)/K(m)) and mannobiose (4.32 k(cat)/K(m)). The enzyme showed transglycosylation and transgalactosylation activities toward cellobiose, lactose and mannooligosaccharides that could produce GOS (galactooligosaccharides) and MOS (maltooligosaccharides). This novel hyperthermostable β-glycosidase may be useful for food and pharmaceutical applications.

A Systematic NMR Determination of α-D-Glucooligosaccharides, Effect of Linkage Type, Anomeric Configuration and Combination of Different Linkages Type on13C Chemical Shifts for the Determination of Unknown Isomaltooligosaccharides

Prebiotic isomaltooligosaccharide preparations contain <TEX>$\alpha$</TEX>-D-glucooligosaccharides comprising isomaltooligosaccharides (IMOs) and non-prebiotic maltooligosaccharides (MOs). They are both glucose oligosaccharides characterized by their degree of polymerization (DP) value (from 2 to <TEX>$\sim$</TEX>10), linkages types and positions (IMOs: <TEX>$\alpha$</TEX>-(1<TEX>$\rightarrow$</TEX>2, 3, 6 and in a lower proportion internal 1<TEX>$\rightarrow$</TEX>4) linkages, MOs: α-(1<TEX>$\rightarrow$</TEX>4) linkages). Their structure is the key factor for their prebiotic potential. In order to determine and elucidate the exact structure of unknown IMOs and MOs, unambiguous assignments of <TEX>$^{13}C$</TEX> and <TEX>$^1H$</TEX> chemical shifts of commercial standards, representative of IMOs and MOs diversity, have been determined using optimized standard one and two-dimensional experiments such as <TEX>$^1H$</TEX> NMR, <TEX>$^{13}C$</TEX> NMR, APT and <TEX>${^1}H-{^1}H$</TEX> COSY, TOCSY, NOESY and <<TEX>$^1H-{^{13}}C$</TEX> heteronuclear HSQC, HSQC-TOCSY, and HMBC. Here we point out the differential effect of substitution by a glucose residue at different positions on chemical shifts of anomeric as well as ring carbons together with the effect of the reducing end configuration for low DP oligosaccharides and diasteroisotopic effect for H-6 protons. From this study, structural <TEX>$^{13}C$</TEX> specific spectral features can be identified as tools for structural analysis of isomaltooligosaccharides.

Dual polarity accurate mass calibration for electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry using maltooligosaccharides

In view of the fact that memory effects associated with instrument calibration hinder the use of many mass-to-charge ( m/ z) ratios and tuning standards, identification of robust, comprehensive, inexpensive, and memory-free calibration standards is of particular interest to the mass spectrometry community. Glucose and its isomers are known to have a residue mass of 162.05282 Da; therefore, both linear and branched forms of polyhexose oligosaccharides possess well-defined masses, making them ideal candidates for mass calibration. Using a wide range of maltooligosaccharides (MOSs) derived from commercially available beers, ions with m/z ratios from approximately 500 to 2500 Da or more have been observed using Fourier transform ion cyclotron resonance mass spectrometry (FT–ICR–MS) and time-of-flight mass spectrometry (TOF–MS). The MOS mixtures were further characterized using infrared multiphoton dissociation (IRMPD) and nano-liquid chromatography/mass spectrometry (nano-LC/MS). In addition to providing well-defined series of positive and negative calibrant ions using either electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI), the MOSs are not encumbered by memory effects and, thus, are well-suited mass calibration and instrument tuning standards for carbohydrate analysis.

STA11, a Chlamydomonas reinhardtii locus required for normal starch granule biogenesis, encodes disproportionating enzyme. Further evidence for a function of alpha-1,4 glucanotransferases during starch granule biosynthesis in green algae.

In Chlamydomonas reinhardtii, the presence of a defective STA11 locus results in significantly reduced granular starch deposition displaying major modifications in shape and structure. This defect simultaneously leads to the accumulation of linear malto-oligosaccharides (MOS). The mutants of STA11 were showed to lack D-enzyme, a plant alpha-1,4 glucanotransferase analogous to the Escherichia coli amylomaltase. We have cloned and characterized both the cDNA and gDNA corresponding to the C. reinhardtii D-enzyme. We now report allele-specific modifications of the D-enzyme gene in the mutants of STA11. These allele-specific modifications cosegregate with the corresponding sta11 mutations, thereby demonstrating that STA11 encodes D-enzyme. MOS production and starch accumulation were investigated during day and night cycles in wild-type and mutant C. reinhardtii cells. We demonstrate that in the algae MOS are produced during starch biosynthesis and degraded during the phases of net polysaccharide catabolism.

Understanding Catalytic Properties and Functions of Maize Starch Synthase Isozymes

We have previously reported the kinetic properties of maize starch synthase I (SSI), SSIIa and SSIIb using glycogen and amylopectin as primer and have determined that these SS enzymes can be distinguished based on their kinetic properties. However, it is not clear how starch synthesis is initiated and what the native primer for SS in vivo is. Therefore, in this study, we determined whether maize SS can catalyze the de novo synthesis of α-glucan and studied how maize SS utilizes potential native primers such as maltooligosaccharides (MOS) and developing native starch granules. Using purified recombinant maize SSI, we provide evidence that maize SSI cannot catalyze the de novo synthesis of α-glucan and the “unprimed activity” may be due to the presence of primers in ADPG. However, maize SSI, SSIIa and SSIIb, can use maltose as a primer, although much less efficiently than MOS with a DP of 3 or greater. All MOSs tested can be elongated by SSI, SSIIa and SSIIb with more than one glucose residues. For the first time we describe here that purified maize SS can further elongate the chains of native starch granule isolated from developing maize waxy endosperm (18 days after pollination). Compared with their kinetic properties in the presence of soluble amylopectin and glycogen, differences in the Vmax and optimum temperature of these three enzymes were ob served in the presence of native starch granules. The differences between SSI, SSIIa and SSIIb in their primer preferences using MOS and native starch granule may provide some clues as to the functions of different SS in starch synthesis.

The Priming of Amylose Synthesis in Arabidopsis Leaves

We investigated the mechanism of amylose synthesis in Arabidopsis leaves using (14)C-labeling techniques. First, we tested the hypothesis that short malto-oligosaccharides (MOS) may act as primers for granule-bound starch synthase I. We found increased amylose synthesis in isolated starch granules supplied with ADP[(14)C]glucose (ADP[(14)C]Glc) and MOS compared with granules supplied with ADP[(14)C]Glc but no MOS. Furthermore, using a MOS-accumulating mutant (dpe1), we found that more amylose was synthesized than in the wild type, correlating with the amount of MOS in vivo. When wild-type and mutant plants were tested in conditions where both lines had similar MOS contents, no difference in amylose synthesis was observed. We also tested the hypothesis that branches of amylopectin might serve as the primers for granule-bound starch synthase I. In this model, elongated branches of amylopectin are subsequently cleaved to form amylose. We conducted pulse-chase experiments, supplying a pulse of ADP[(14)C]Glc to isolated starch granules or (14)CO(2) to intact plants, followed by a chase period in unlabeled substrate. We detected no transfer of label from the amylopectin fraction to the amylose fraction of starch either in isolated starch granules or in intact leaves, despite varying the time course of the experiments and using a mutant line (sex4) in which high-amylose starch is synthesized. We therefore find no evidence for amylopectin-primed amylose synthesis in Arabidopsis. We propose that MOS are the primers for amylose synthesis in Arabidopsis leaves.