Untersuchungen am Phloemexsudat von Cucurbita pepo L.

Summary Tests for enzymes of gluconeogenesis and of the synthesis and degradation of sucrose and polysaccharides have been carried out in the phloem exudate of Cucurbita pepo. All the enzymes which are necessary for the synthesis of sucrose and polysaccharides from metabolites of the citric acid cycle were found to be present in the exudate, except phosphoenolpyruvate carboxykinase. The polysaccharide synthetase was found to exhibit higher activity with glycogen (which is an unnatural polysaccharide in higher plants) than with starch. In addition, polysaccharide synthetase activity could be increased remarkably with 2 mM glucose-6-phosphate and glycogen as primer. Among the enzymes which catabolize sucrose and polysaccharides (phosphorylase, invertase, sucrose phosphorylase), only sucrose phosphorylase showed activity.     

Regulation of glucose metabolism and cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent β-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only β-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only β-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.     

Effect of cetyltrimethylammonium bromide on the activity of particulate starch synthetase from potato tuber

The action of some detergents on the incorporation of glucose from uridine diphosphate glucose or adenosine diphosphate glucose into the potato tuber starch grain was studied. It was found that the cationic detergent, cetyltrimethylammonium bromide, produces a rapid binding of both sugar nucleotides to the grain and a great increase in the incorporation of glucose into the polysaccharide. Kinetic constants of starch synthetase are also modified, there being an affinity increase for both sugar nucleotides. Neutral detergents are without effect and anionic detergents are inhibitors.     

Biosynthesis of Glycogen and Starch in Cryptococcus laurentii

Cells of Cryptococcus laurentii, when grown in liquid culture on 2% glucose close to neutral pH, showed glycogen granules throughout the cytoplasm. Glycogen levels of C. laurentii cells reached maximal levels just before onset of stationary phase. Concomitantly, a sharp rise in total and specific activity of glycogen synthetase was observed. Conversely, glycogen phosphorylase reached its highest specific activity approximately 3 hr after the glycogen peaked and remained high until most of the endogenous glycogen was utilized. Uridine diphosphoglucose pyrophosphorylase activity was always an order of magnitude higher than glycogen synthetase during log phase, but fell off rapidly after the cells reached stationary growth. Kinetic properties of the glycogen synthetase showed that the enzyme is always activated by glucose-6-phosphate, although the degree of activation by glucose-6-phosphate was found to be somewhat variable. The accelerated uptake of glucose commencing with the onset of stationary phase is explained by the rapid formation of extracellular acidic polysaccharide, which continues as long as there is glucose in the medium. In cells grown at pH 3.4, where no detectable extracellular acidic polysaccharide was formed, glucose uptake drastically declined when the cells reached stationary phase. These cells also contained glycogen-like granules in the cytoplasm. The evidence presented indicates that these granules are in fact glycogen, and that its structure does not resemble that of the starch excreted by cells grown at acidic pH.     

Biosynthesis of starch. Formation of a glucoproteic acceptor by a potato non-sedimentable preparation.

1. A non-sedimentable fraction of potato tuber has been found to catalyze [14C]glucose transfer from [14C]glucose 1-phosphate to an endogenous proteic acceptor in the absence of added primer. This transfer is activated by Mn2+. 2. The labeled glucosylated product formed is trichloroacetic acid insoluble and sensitive to proteolytic and amylolytic digestions. It appears to be a glucoprotein with glucosyl chains bound to the peptide portion of the molecule through an unknown linkage. 3. The carbohydrate portion of the glucoprotein can be released by prolonged incubations with the enzymatic preparation, and becomes in turn, trichloroacetic acid soluble and alcohol precipitable. 4. Both products, the glucoprotein as well as the alpha-1,4-glucan that seems to arise from the enzymatic cleavage of the former, can be used as primers by the transglucosylating system with ADP[14C]glucose, UDP[14C]glucose or GDP[14C]glucose as glucosyl donors. The results presented in this paper are the first demonstration of soluble glucosyl transferases with the same glucose donor specificity to that of the particulate starch synthetase. 5. This report presents further evidence in favor of the assumption of a glucoproteic intermediate in alpha-a,4-glucan synthesis initiation.     

Differential inhibition of branching enzyme in a morphological mutant and in wild type Neurospora. Influence of carbon source in the growth medium.

1. A morphological mutant of Neurospora crassa, smco 9, (R2508) that exhibits colonial morphology when grown on sucrose or on maltose, showed a partial reversal of this morphology toward that of the wild type when it was grown on potato starch or on isomaltose. 2. A common feature of both potato starch and isomaltose is the presence of alpha-1, 6 glucosidic linkages. This suggested that these morphological effects might be due to differences in alpha-1,4 glucan: alpha-1,4 glucan 6 glycosyltransferase, (EC 2.4.1.18) commonly known as "the branching enzyme". 3. The branching enzyme was purified from wild type, Neurospora crassa, and from the semicolonial mutant, R2508, both grown on sucrose or on potato starch. It has a molecular weight of 140,000 as estimated by gel filtration on a Bio Gel A 1.5 m column. This enzyme plus phosphorylase a in an unprimed reaction catalyzes the synthesis of a branched polysaccharide in vitro. 4. No branching enzyme activity was apparent in extracts of the mutant R2508, grown on potato starch until a thermolabile inhibitor was removed by fractionation on a DEAE column. 5. This inhibitor has a molecular weight greater than 100,000 as estimated on a P-100 polyacrylamide gel column. The specificity of the inhibitor is not absolute in that it inhibits glycogen synthetase in addition to the branching enzyme in Neurospora.     

Characterization of starch breakdown in the intact spinach chloroplast

Starch degradation with a rate of 1 to 2 microgram-atom carbon per milligram chlorophyll per hour was monitored in the isolated intact spinach (Spinacia oleracea) chloroplast which had been preloaded with ¹⁴C-starch photosynthetically from ¹⁴CO₂. Starch breakdown was dependent upon inorganic phosphate and the ¹⁴C-labeled intermediates formed were principally those of the Embden-Meyerhof pathway from glucose phosphate to glycerate 3-phosphate. In addition, isotope was found in ribose 5-phosphate and in maltose and glucose. The appearance of isotope in the intermediates of the Embden-Meyerhof pathway but not in the free sugars was dependent upon the inorganic phosphate concentration. Dithiothreitol shifted the flow of ¹⁴C from triose-phosphate to glycerate 3-phosphate. Iodoacetic acid inhibited starch breakdown and caused an accumulation of triose-phosphate. This inhibition of starch breakdown was overcome by ATP. The inhibitory effect of ionophore A 23187 on starch breakdown was reversed by the addition of magnesium ions. The formation of maltose but not glucose was impaired by the ionophore. The inhibition of starch breakdown by glycerate 3-phosphate was overcome by inorganic phosphate. Fructose 1,6-bisphosphate and ribose 5-phosphate did not affect the rate of polysaccharide metabolism but increased the flow of isotope into maltose. Starch breakdown was unaffected by the uncoupler (trifluoromethoxyphenylhydrazone), electron transport inhibitors (rotenone, cyanide, salicylhydroxamic acid), or anaerobiosis. Hexokinase and the dehydrogenases of glucose 6-phosphate and gluconate 6-phosphate were detected in the chloroplast preparations. It was concluded (a) that chloroplastic starch was degraded principally by the Embden-Meyerhof pathway and by a pathway involving amylolytic cleavage; (b) ATP required in the Embden-Meyerhof pathway is generated by substrate phosphorylation in the oxidation of glyceraldehyde 3-phosphate to glycerate 3-phosphate; and (c) the oxidative pentose phosphate pathway is the probable source of ribose 5-phosphate.     

Fluoride-Induced Inhibition of Starch Biosynthesis in Developing Potato, Solanum tuberosum L., Tubers Is Associated with Pyrophosphate Accumulation

Pretreatment of discs excised from developing tubers of potato (Solanum tuberosum L.) with 10 millimolar sodium fluoride induced a transient increase in 3-phosphoglycerate content. This was followed by increases in triose-phosphate, fructose 1,6-bisphosphate and hexose-phosphate (glucose 6-phosphate + fructose 6-phosphate + glucose 1-phosphate). The effect of fluoride is attributed to an inhibition of glycolysis and a stimulation of triose-phosphate recycling (the latter confirmed by the pattern of ¹³C-labeling [NMR] in sucrose when tissue was supplied with [2-¹³C]glucose). Fluoride inhibited the incorporation of [U-¹⁴C] glucose, [U-¹⁴C]sucrose, [U-¹⁴C]glucose 1-phosphate, and [U-¹⁴C] glycerol into starch. The incorporation of [U-¹⁴C]ADPglucose was unaffected. Inhibition of starch biosynthesis was accompanied by an almost proportional increase in the incorporation of ¹⁴C into sucrose. The inhibition of starch synthesis was accompanied by a 10-fold increase in tissue pyrophosphate (PPi) content. Although the subcellular localization of PPi was not determined, a hypothesis is presented that argues that the PPi accumulates in the amyloplast due to inhibition of alkaline inorganic pyrophosphatase by fluoride ions.     

Control of amino sugar metabolism in Escherichia coli and isolation of mutants unable to degrade amino sugars

1. Growth of Escherichia coli on glucosamine results in an induction of glucosamine 6-phosphate deaminase [2-amino-2-deoxy-d-glucose 6-phosphate ketol-isomerase (deaminating), EC 5.3.1.10] and a repression of glucosamine 6-phosphate synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16); glucose abolishes these control effects. 2. Growth of E. coli on N-acetylglucosamine results in an induction of N-acetylglucosamine 6-phosphate deacetylase and glucosamine 6-phosphate deaminase, and in a repression of glucosamine 6-phosphate synthetase; glucose diminishes these control effects. 3. The synthesis of amino sugar kinases (EC 2.7.1.8 and 2.7.1.9) is unaffected by growth on amino sugars. 4. Glucosamine 6-phosphate synthetase is inhibited by glucosamine 6-phosphate. 5. Mutants of E. coli that are unable to grow on N-acetylglucosamine have been isolated, and lack either N-acetylglucosamine 6-phosphate deacetylase (deacetylaseless) or glucosamine 6-phosphate deaminase (deaminaseless). Deacetylaseless mutants can grow on glucosamine but deaminaseless mutants cannot. 6. After growth on glucose, deacetylaseless mutants have a repressed glucosamine 6-phosphate synthetase and a super-induced glucosamine 6-phosphate deaminase; this may be related to an intracellular accumulation of acetylamino sugar that also occurs under these conditions. In one mutant the acetylamino sugar was shown to be partly as N-acetylglucosamine 6-phosphate. Deaminaseless mutants have no abnormal control effects after growth on glucose. 7. Addition of N-acetylglucosamine or glucosamine to cultures of a deaminaseless mutant caused inhibition of growth. Addition of N-acetylglucosamine to cultures of a deacetylaseless mutant caused lysis, and secondary mutants were isolated that did not lyse; most of these secondary mutants had lost glucosamine 6-phosphate deaminase and an uptake mechanism for N-acetylglucosamine. 8. Similar amounts of (14)C were incorporated from [1-(14)C]-glucosamine by cells of mutants and wild-type growing on broth. Cells of wild-type and a deaminaseless mutant incorporated (14)C from N-acetyl[1-(14)C]glucosamine more efficiently than from N[1-(14)C]-acetylglucosamine, incorporation from the latter being further decreased by acetate; cells of a deacetylaseless mutant showed a poor incorporation of both types of labelled N-acetylglucosamine.     

Some Properties of Potato Tuber UDPGd-fructose-2-glucosyltransferase (E.C. 2.4.1.14) and UDPGd-fructose-6-phosphate-2-glucosyltransferase (E.C. 2.4.1.13)

Sucrose and sucrose 6-phosphate synthetase were isolated from potato tubers, partially purified and their properties studied. The sucrose synthetase showed optimum activity at 45° and was inhibited competitively by ADP and some phenolic glucosides. The Ki′s for these inhibitors were determined. Mg²⁺ was found to activate this enzyme. Activity toward UDP-glucose or ADP-glucose formation was measured. The optimum conditions for sucrose and UDP-glucose formation were found to differ. The specificity for the glucosyl donor and acceptor were determined.

The optimum conditions for sucrose 6-phosphate synthetase activity were studied. This enzyme was not inhibited by either ADP or phenolic glucosides; UDP-glucose was the only glucosyl donor for sucrose 6-phosphate formation.

     

Evidence that glucose 6-phosphate is imported as the substrate for starch synthesis by the plastids of developing pea embryos

The aim of this work was to determine in what form carbon destined for starch synthesis crosses the membranes of plastids in developing pea (Pisum sativum L.) embryos. Plastids were isolated mechanically and incubated in the presence of ATP with the following ¹⁴C-labelled substrates: glucose, fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate. Glucose 6-phosphate was the only substrate that supported physiologically relevant rates of starch synthesis. Incorporation of label from glucose 6-phosphate into starch was dependent upon the integrity of the plastids and the presence of ATP. The rate of incorporation approached saturation at a glucose 6-phosphate concentration of less than 1 mM. It is argued that glucose 6-phosphate is likely to enter the plastid as the source of carbon for starch synthesis in vivo.Abbreviations ADPG PPase ADP-glucose pyrophosphorylase - DHAP dihydroxyacetone phosphate     

Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens.

The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.     

The binding of substrates and modifiers to glucosamine synthetase

1. The binding of substrates and effectors to glucosamine synthetase (l-glutamine-d-fructose 6-phosphate aminotransferase, EC 2.6.1.16) was studied by using the ligand to alter the denaturation rate of the enzyme. The free enzyme bound fructose 6-phosphate, glucose 6-phosphate and UDP-N-acetylglucosamine, but not glutamine, AMP or UTP. Glucose 6-phosphate and AMP increased the binding of UDP-N-acetylglucosamine whereas UTP decreased the interaction between the enzyme and the feedback inhibitor. UDP-N-acetylglucosamine induced a glutamine-binding site on the enzyme. 2. Selective thermal or chemical denaturation revealed that the UDP-N-acetylglucosamine-binding site was not located at the catalytic site. The UTP site could not be distinguished from that for the nucleotide sugar. The AMP- and glucose 6-phosphate-binding sites were distinct from the catalytic and feedback-inhibitor-binding sites. 3. The specificity of the glutamine-binding site was investigated by using a series of potential analogues. 4. A model is proposed for the action of the effectors and the mechanism of the reaction discussed in kinetic and chemical terms.     

The uptake and metabolism of glucose, maltose and starch by the rumen ciliate Epidinium ecaudatum caudatum.

[14C]Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C]glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccaride was poor. Hexokinase, phosphoglucomutase, alpha-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.     

Effects of isoniazid on the composition of mycobacteria, with particular reference to soluble carbohydrates and related substances

1. When growing Mycobacterium tuberculosis BCG was exposed to 0.5-10mug. of isoniazid/ml. there was intracellular accumulation of soluble carbohydrate, combined phosphate and substances absorbing at 260mmu. Yellow pigments were formed when modified Sauton medium was used, but not with Proskauer & Beck medium. These processes were apparent after 1hr. but were more marked after about 6hr. These effects were not found with an isoniazid-resistant strain. 2. After 6hr. exposure of the sensitive strain to 10mug./ml. there was little change in the amounts (per g. of insoluble nitrogen) of total lipid, glycolipid, RNA, DNA or of carbohydrate in the nucleic acid fractions. 3. The major accumulation was of alphaalpha'-trehalose. There was also accumulation of glucose 6-phosphate, glucose 1-phosphate, fructose 6-phosphate, trehalose 6-phosphate (tentatively identified), a polysaccharide containing only glucose, and an oligosaccharide containing glucose and glucose 6-phosphate, but not of glycerol and glycerol 3-phosphate. The u.v.-absorbing materials appeared to be nucleotide sugar derivatives. 4. In Mycobacterium smegmatis a similar accumulation of trehalose occurred on exposure to isoniazid, but there was little accumulation of other compounds. 5. No evidence could be found that isoniazid specifically affected the oxidation of glycerol or glycerol 3-phosphate. 6. It is suggested that the primary action of isoniazid on mycobacteria may be partial inhibition of a reaction in some central area of metabolism, such as glycolysis.     

Unusual starch degradation pathway via cyclodextrins in the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324

The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, alpha-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly beta-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.     

Stimulation of pentose phosphate pathway dehydrogenase enzyme activities in ethionine-treated mice

1. Mice treated with ethionine (intraperitoneally, 5mg./day for 4 days or 10mg./day for 3 days) showed a profound loss of hepatic glycogen, a decrease of glycogen synthetase activity, a development of hypoglycaemia, a two- to five-fold increase in the activity of glucose 6-phosphate dehydrogenase but no change in 6-phosphogluconate dehydrogenase and an earlier manifestation of the solubilization of phosphorylase as compared with glycogen synthetase. The administration of ATP did not prevent these effects. 2. During the early post-injection period (2-3 days) there was a further enhancement of the activity of glucose 6-phosphate dehydrogenase (tenfold) in the liver and a clear elevation of 6-phosphogluconate dehydrogenase activity (twofold). Subsequently, the glycogen concentration was restored, followed by an earlier reassociation of glycogen particle with phosphorylase than with glycogen synthetase, along with a disappearance of ethionine effect at about the eighteenth day. 3. Glucose 6-phosphate dehydrogenase from both control and ethionine-treated animals showed a marked preference for glucose 6-phosphate as substrate rather than for galactose 6-phosphate, whose rate of oxidation was only 10% of that of the glucose 6-phosphate. 4. Since actinomycin D, puromycin, 5-fluorouracil and dl-p-fluorophenylalanine failed to block the ethionine-enhanced glucose 6-phosphate dehydrogenase activity, the possibility that new enzyme protein synthesis is responsible for the effect is doubtful.     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

Mass spectrometric quantification of the relative amounts of C6 and C3 position phosphorylated glucosyl residues in starch

The quantification of phosphate bound to the C6 and C3 positions of glucose residues in starch has received increasing interest since the importance of starch phosphorylation for plant metabolism was discovered. The method described here is based on the observation that the isobaric compounds glucose-6-phosphate (Glc6P) and glucose-3-phosphate (Glc3P) exhibit significantly different fragmentation patterns in negative ion electrospray tandem mass spectrometry (MS/MS). A simple experiment involving collision-induced dissociation (CID) MS² spectra of the sample and the two reference substances Glc3P and Glc6P permitted the quantification of the relative amounts of the two compounds in monosaccharide mixtures generated by acid hydrolysis of starch. The method was tested on well-characterized potato tuber starch. The results are consistent with those obtained by NMR analysis. In contrast to NMR, however, the presented method is fast and can be performed on less than 1 mg of starch. Starch samples of other origins exhibiting a variety of phosphorylation degrees were analyzed to assess the sensitivity and robustness of the method.