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First published online July 30, 2004; 10.1104/pp.104.041301 Plant Physiology 135:2068-2077 (2004) © 2004 American Society of Plant Biologists Phosphorylation of Transitory Starch Is Increased during Degradation1,[w]Plant Physiology, Institute of Biochemistry and Biology (G.R., A.S., N.E., M.S.), and Center for Biopolymers (S.H.), University of Potsdam, D–14476 Golm, Germany
The starch excess phenotype of Arabidopsis mutants defective in the starch phosphorylating enzyme glucan, water dikinase (EC 2.7.9.4) indicates that phosphorylation of starch is required for its degradation. However, the underlying mechanism has not yet been elucidated. In this study, two in vivo systems have been established that allow the analysis of phosphorylation of transitory starch during both biosynthesis in the light and degradation in darkness. First, a photoautotrophic culture of the unicellular green alga Chlamydomonas reinhardtii was used to monitor the incorporation of exogenously supplied 32P orthophosphate into starch. Illuminated cells incorporated 32P into starch with a constant rate during 2 h. By contrast, starch phosphorylation in darkened cells exceeded that in illuminated cells within the first 30 min, but subsequently phosphate incorporation declined. Pulse-chase experiments performed with 32P/31P orthophosphate revealed a high turnover of the starch-bound phosphate esters in darkened cells but no detectable turnover in illuminated cells. Secondly, leaf starch granules were isolated from potato (Solanum tuberosum) plants grown under controlled conditions and glucan chains from the outer granule layer were released by isoamylase. Phosphorylated chains were purified and analyzed using high performance anion-exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry. Glucans released from the surface of starch granules that had been isolated from darkened leaves possessed a considerably higher degree of phosphorylation than those prepared from leaves harvested during the light period. Thus, in the unicellular alga as well as in potato leaves, net starch degradation is accompanied with an increased phosphorylation of starch.
Starch is the predominant carbohydrate reserve in plants and is deposited as semicrystalline particles. One can distinguish reserve starch in storage organs and transitory starch in photosynthetic organs. Whereas reserve starch can be stored over months or years, transitory starch is normally accumulated during the day and degraded in the following dark period. Starch consists of the two Glc polymers, amylose and amylopectin. Amylose comprises predominantly linear chains of  -1,4-linked Glc residues, whereas amylopectin exists as a branched -1,4:-1,6 D-glucan polymer. Amylopectin accounts for about 75% of the starch weight in reserve starch (Ball et al., 1998 ) and typically more than 90% in transitory starch (Zeeman et al., 2002).
Amylopectin contains small amounts of phosphate monoesters (Hizukuri et al., 1970
Under in vitro conditions this reaction leads to the formation of glucan-bound Glc6P and Glc3P residues in a ratio of approximately 2:1, which is also found in native starch (Tabata and Hizukuri, 1971
The physiological relevance of starch phosphorylation is not yet fully understood. However, the analyses of plants with reduced GWD activity indicate that the phosphorylation of starch affects its in vivo degradability. Starch breakdown is strongly impaired in GWD antisense potato plants and in the GWD-deficient starch-excess 1 (sex1) mutants of Arabidopsis (Lorberth et al., 1998
Nielsen et al. (1994)
In Vivo Starch Phosphorylation in Chlamydomonas
Genomic analysis provides evidence for the existence of GWD-like sequences in Chlamydomonas (Mikkelsen et al., 2004 Synthesis and degradation of starch were monitored in labeling experiments using NaH14CO3. In illuminated cells, the rate of starch synthesis was high, ranging from 93 to 133 µmol C (mg chlorophyll [Chl])–1 h–1 in four independent experiments. No incorporation of 14C into starch was detectable in darkened cells. If starch was prelabeled with 14C in the light and the cells were then transferred to darkness, we consistently observed a rapid decrease in the labeled starch fraction (Fig. 1). This indicates that the darkened cells rapidly initiate starch mobilization.
Phosphate incorporation into starch was monitored by adding 32P-orthophosphate to the cell suspension. Following incubation, cells were quickly lysed in 2% (w/v) SDS, and insoluble compounds were pelleted by centrifugation. After several washing steps, starch was solubilized using a mixture of -amylase and amyloglucosidase. Following this treatment, no particulate material was detectable, and, therefore, the pellets almost exclusively consisted of starch. The algal cells incorporated phosphate into starch during both synthesis and degradation (Fig. 2A). Within the first 30 min of labeling, starch phosphorylation in darkened cells exceeded that in illuminated cells. However, in darkness, net phosphate incorporation markedly slowed down with time. Maximal 32P content was reached after 90 min. By contrast, illuminated cells incorporated phosphate into starch with an essentially constant rate during the 2-h incubation period. Under the experimental conditions used, the phosphate uptake into the cells was slightly higher in the light than in darkness. During 40 min, illuminated and darkened cells took up 1.27 and 1.06 µmol Pi (mg Chl)–1, respectively.
The turnover of the starch-bound phosphate was studied by using pulse-chase experiments. After 1 h of incubation with 32P-orthophosphate, a 500-fold excess of nonlabeled phosphate was added. The amount of radioactivity in the starch was analyzed 20 and 40 min after the chase (Fig. 2B). No turnover of the starch-bound phosphate is detectable during starch synthesis in the light. The 32P labeling of the starch even slightly increased during the chase. Presumably, this is due to a delayed decrease in the intracellular specific radioactivity (e.g. in the plastidial ATP pool). By contrast, in darkened algae, more than 50% of the starch-associated radioactivity was lost within 20 min after the chase. This demonstrates that starch phosphorylation during starch breakdown is transient, and the phosphate esters formed underlie a rapid turnover. Thus, starch phosphorylation during degradation cannot be calculated from the net incorporation of radioactivity into starch. Based on the data shown in Figure 2, one can roughly estimate that the rate of phosphorylation during starch breakdown exceeds that during synthesis by (at least) a factor of two. The data obtained by an independent labeling and pulse-chase experiment are included as supplemental material (available at www.plantphysiol.org). From the mean rates of starch synthesis and starch phosphorylation in the light (average of two independent experiments), we calculated an esterification of approximately 0.2 nmol P/µmol Glc. Probably, this value is an underestimation, since the specific radioactivity of the plastidic ATP pool is likely to be (at least temporarily) lower than that of the orthophosphate supplied to the cells. However, this value is similar to starch phosphate contents of leaf starch (Blennow et al., 2000 ; Yu et al., 2001).
Starch Phosphorylation in Chlamydomonas Occurs at C6 and C3 Position of the Glc Units
Starch Phosphorylation in Potato Leaves Intact higher plants are not suited for short-term labeling studies using phosphate isotopes, thus an alternative experimental system was required to investigate in vivo starch phosphorylation in potato leaves. It is reasonable to assume that most of the starch phosphorylation occurring during net starch degradation (compare with Fig. 2) takes place at the surface of the starch granule. As the phosphate level at the starch granule surface is likely to reflect the actual phosphorylating activity under various physiological conditions, we decided to analyze the glucan chains exposed to the starch granule surface.
Release of Glucan Chains from the Outer Layer of Leaf Starch Granules
Starch granules were prepared from potato leaves that had been harvested at different times during the day/night cycle. Granules were isolated under denaturating conditions to remove granule surface-associated proteins and to prevent enzymatic modification of the starch during extraction. The granules were then treated with isoamylase for 20 min, and the glucan chains released were analyzed using HPAEC-PAD (Fig. 5). While the total amount of released glucans was very similar in the different samples, two qualitative differences became evident. First, the proportion of the very short chains (DP 3, DP 4, and especially DP 5) strongly increased upon darkening of the leaves. Second, a small late eluting peak (Fig. 5, marked by an arrow) was clearly present in samples from darkened leaves but hardly detectable in those from illuminated leaves. The same characteristic differences were observed in an independent experiment (data not shown). These alterations are restricted to glucan chains at the granule surface. We extracted starch from darkened leaves and compared the patterns of glucan chains released by isoamylase from either intact granules (Fig. 6A) or solubilized starch (Fig. 6B). Solubilized starch is completely debranched by the enzyme. In contrast with the sample derived from intact granules (Fig. 6A), the very short chains and the late eluting peak were hardly detectable in the chain length distribution profile of completely debranched starch (Fig. 6B).
The Phosphorylation Level of the Granule Surface Is Increased during Starch Breakdown In order to purify phosphorylated side chains, the compounds released by isoamylase were subjected to anion-exchange chromatography using Q Sepharose. Uncharged chains were removed from the column by extensive washing with water. Subsequently, charged chains were eluted using a mixture of NaCl and HCl and were then analyzed by HPAEC-PAD (Fig. 7). In samples derived from darkened leaves, two peaks were eluted between 20 and 25 min. These peaks were hardly detectable in samples prepared from illuminated leaves (Fig. 7). This result was confirmed in an independent experiment (data not shown). When the charged chains purified from darkened leaves (4 h D, compare with Fig. 7) were cochromatographed with the total glucan pool released from granules that had been prepared from illuminated leaves (8 h L, compare with Fig. 5), it became evident that the phosphorylated glucans comprise the shallow late peak (compare with Figs. 5 and 6, marked by arrows) present in the nonfractioned chains in samples from darkened leaves.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis confirmed that the glucans purified by anion-exchange chromatography (compare with Fig. 7) are phosphorylated oligosaccharides (Fig. 8). Singly phosphorylated glucan chains ranging from DP 7 to DP 23 (Fig. 8A) were detected in the sample (one-step elution) derived from intact starch granules from darkened leaves (4 h D). A further resolution of the phosphorylated glucans into two fractions (peaks 1 and 2; compare with Fig. 7) by stepwise elution from the anion-exchanger revealed that side chains carrying two phosphate groups were also present. The compounds eluting in peak 2 (compare with Fig. 7) consist of doubly phosphorylated oligosaccharides ranging from DP 14 to DP 32 (Fig. 8B), whereas the material eluting in peak 1 comprises the monophosphorylated glucans. The mass spectra obtained closely resembled that depicted in Figure 8A (data not shown). Thus, in darkened leaves, the phosphate content of the starch granule surface is strongly increased. It should be noted that this increase was poorly reflected on the total starch level. This conclusion was reached when aliquots from the starch granule preparations analyzed in Figures 5, 7, and 8 were subjected to acid hydrolysis and the total Glc6P to Glc ratio was determined (Table I). The total starch phosphate content was only moderately elevated in the starch samples prepared from darkened leaves.
Starch Granules from Darkened Leaves Are Better Substrates for GWD in Vitro Compared with Granules from Illuminated Leaves The data shown in Figure 7 strongly indicate that, similar to the results obtained in Chlamydomonas, the level of starch phosphorylation is higher during starch breakdown than during starch biosynthesis. However, in principle an alternative explanation is possible: If neutral glucan chains are degraded more rapidly than phosphorylated ones, the latter would be expected to increase relative to the uncharged chains. Two lines of evidence indicate that this is very unlikely to be relevant. First, in vitro phosphorylation of the granule samples with recombinant GWD revealed that starch granules extracted from darkened leaves were phosphorylated with an up to 7-fold higher rate compared with granules from illuminated leaves (Fig. 9). This increase was not caused by altered surface-to-mass ratios because the mean granule diameters were comparable in the different starch samples (revealed by size distribution analysis using a Coulter Counter; data not shown). Second, in vivo the amount of granule surface-associated GWD strongly increased under conditions of starch degradation in potato leaves, whereas the total amount of GWD, which is predominantly soluble, is unchanged (Ritte et al., 2000a ). This result was confirmed using starch and soluble protein prepared under nondenaturating conditions from aliquots of the leaf samples that had been used for the analysis of the surface-bound glucan chains (data not shown).
In photosynthesizing Chlamydomonas cells, starch is phosphorylated concurrently with starch biosynthesis without detectable turnover. Likewise, in potato leaves, starch phosphorylation occurs also during biosynthesis, as indicated by the similar levels of starch-bound Glc6P in light and darkness (Table I). Thus, transitory starch is phosphorylated during synthesis, as it has previously been documented for reserve starch in potato amyloplasts (Nielsen et al., 1994 ; Wischmann et al., 1999). However, here we show that during breakdown of transitory starch, phosphorylation occurs at an even higher rate. Phosphorylation of Chlamydomonas starch under conditions of net starch breakdown is transient as it is subjected to a substantial turnover. Probably, this is due to the fact that both phosphorylation and degradation take place at the granule surface.
Elevated starch phosphorylation during breakdown of the granule implies that GWD plays an active role during starch degradation. In leaves of Arabidopsis, potato, and pea, the total amounts of the GWD protein are apparently unchanged during light and darkness (Ritte et al., 2000a
In addition to modifications of the granule surface, posttranslational modification of GWD might also be involved in the increased glucan phosphorylation during starch breakdown. In leaves, the regulation of starch synthesis involves redox modulation of ADP-Glc pyrophosphorylase activity in response to light and sugars (Hendriks et al., 2003
Our analyses of intact potato leaf starch granules using isoamylase demonstrate that the outer layer of the granules differs from the remainder. This is indicated by the kinetics of isoamylase activity using intact starch granules (Fig. 4). Possibly, the outermost layer displays a lower level of crystallinity and is thus more easily attacked by the enzyme. Furthermore, the distribution profile of glucan chains released by isoamylase from intact granules or solubilized granules, respectively, differs (Fig. 6). Nielsen et al. (2002)
When the surface phosphate levels are compared with the total starch phosphate contents, as determined following hydrolysis of complete granules, it is obvious that increased surface phosphorylation does not necessarily lead to a significant increase in total starch phosphate. Thus, the overall starch phosphate content mainly reflects starch phosphorylation during synthesis, and changes occurring at the surface during degradation are probably too small to significantly alter the total phosphate content of starch. GWD seems to be ubiquitous in higher plants and green algae (Ritte et al., 2000b
Chemicals and Enzymes
[
The cell wall deficient mutant cw15 (Hyams and Davies, 1972 Potato plants (Solanum tuberosum L. cv Desiree) were cultivated in a growth cabinet under controlled conditions (14 h light, approximately 250 µmol quanta m–2 s–1 [20°C]; 10 h dark [16°C]). Leaves from plants in the early flowering stage were analyzed.
Following P-starvation, cells were harvested by centrifugation (10 min at 1,000g, 4°C) and suspended in fresh P-starvation medium to give an OD800 of 2. The cell suspension (0.5 mL each) was transferred into 1.5-mL microtubes. Labeling experiments were started by adding 25 µL of a mixture of NaHCO3 (final concentration 20 mM) and sodium phosphate (final concentration 40 µM). To monitor synthesis or phosphorylation of starch, 0.25 µCi NaH14CO3 or 3 to 6 µCi 32P-orthophosphate were included in this mixture. The tubes were then agitated on a rotating wheel in the light (170 µmol m–2 s–1) or in darkness (<2 µmol m–2 s–1) at 22°C as stated. In the pulse-chase experiments, 50 µL of 230 mM sodium phosphate, pH 7.2, or 50 µL of a mixture of 230 mM sodium phosphate, pH 7.2, and 57.5 mM NaHCO3 were added to cell suspensions (volume = 525 µL) that had been incubated with 32P (see above) for 1 h in darkness or light, respectively. Subsequently, cells were further incubated as stated. Following incubation, cells were lysed by adding one-fifth volume of a 10% (w/v) SDS solution. In control samples, SDS was added prior to the addition of the radioactivity. Following centrifugation for 5 min at 15,000g, the supernatant was discarded and the pellet was washed three times in 0.9 mL of 2% (w/v) SDS, once in 10 mM sodium phosphate, pH 7.2, and two times in 50 mM sodium acetate, pH 5, 5 mM CaCl2. Each washing step was performed for at least 20 min under agitation. The starch was recovered by centrifugation as above. To solubilize the starch, the pellets were resuspended in 0.2 mL of 50 mM sodium acetate, pH 5, 5 mM CaCl2 containing 10 units amyloglucosidase and 140 units
For the analysis of starch granule-associated proteins, starch was extracted from potato leaves according to Ritte et al. (2000a)
For intact starch granules, granules were suspended in 5 mM sodium acetate, pH 4.0, as indicated, and isoamylase (0.25 unit/mg starch) was added. Following incubation at 40°C as stated, the starch granules were pelleted by centrifugation (5 min, 20,000g). The supernatant was collected and heated (5 min, 95°C). For solubilized granules, 2 mg starch were solubilized by adding 375 µL of 75 mM NaOH and incubation for 2 h at 95°C. Following neutralization with 2 N HCl, 100 mM sodium acetate, pH 4.0, was added to give a final concentration of 5 mM. The solubilized starch was debranched with 5 units isoamylase for 30 min at 40°C.
To measure the amount of glucosyl residues present in starch or released from starch granules by isoamylase, starch granules or aliquots of the solubilized glucan chains were hydrolyzed in 1 N HCl for 2 h at 100°C. Following neutralization with KOH, the amount of Glc was assayed according to Waffenschmidt and Jaenicke (1987)
Minicolumns containing 200 µL of Q-Sepharose-FF (Amersham Biosciences, Freiburg, Germany) were prepared using 1-mL plastic pipette tips. A filter paper and a thin layer of glass beads (212–300 microns; Sigma, St. Louis) served as frit to prevent leakage of the Sepharose. The isolation of phosphorylated glucans is based on the method described by Viksø-Nielsen et al. (1998)
HPAEC-PAD analysis was performed as described (Ritte et al., 2000b
Purified phospho-oligosaccharides were dialyzed against water using Spectra/Por Float-A-Lyzer membranes (Spectrum, Breda, The Netherlands) with a cutoff pore size of 500 D. One hundred microliters of sample were then concentrated under vacuum to approximately 10 µL. An aliquot of the concentrated sample (0.5 µL) was mixed on the target with 0.7 µL of a matrix solution containing 2,5-dihydroxybenzoic acid (15 mg mL–1) dissolved in 30% (v/v) aqueous ethanol and was dried under a gentle stream of air. Mass spectra were recorded on a Bruker Reflex II (Bruker Daltonik, Bremen, Germany) in the positive ion mode. For ionization, a nitrogen laser (337 nm, 3 ns pulse width, 3 Hz) was used. For optimization of the mass spectra, the laser was aimed either at the central area of the sample or at the outmost edge of the crystal rim. All spectra were measured in the reflector mode using external calibration.
GWD from potato was heterologously expressed in Escherichia coli and was subsequently purified as described (Ritte et al., 2002
The size distribution of potato leaf starch granules was analyzed using a Multisizer TM3 Coulter Counter (Beckman Coulter) equipped with a 30-µm aperture. Calibration was performed using latex particles of defined size.
Chlorophyll was determined according to Arnon (1949)
We thank Kerstin Pusch for skillful technical assistance and Silke Gopp for plant cultivation. Received February 20, 2004; returned for revision May 5, 2004; accepted May 17, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft (grant nos. SFB–429–TP–B2 to M.S. and TP–B7 to G.R.). 
[w] The online version of this article contains Web-only data. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.041301. * Corresponding author; e-mail ritte{at}rz.uni-potsdam.de; fax 49–331–977–2512.
Arnon DJ (1949) Copper enzymes in isolated chloroplasts. Plant Physiol 24: 1–15 Ball SG, van de Wal MHBJ, Visser RGF (1998) Progress in understanding the biosynthesis of amylose. Trends Plant Sci 3: 462–467[CrossRef] Blennow A, Bay-Schmidt MA, Wischmann B, Olsen CE, Møller BL (1998) The degree of starch phosphorylation is related to chain length distribution of neutral and phosphorylated chains of amylopectin. Carbohydr Res 307: 45–54[CrossRef] Blennow A, Engelsen SB, Munck L, Møller BL (2000) Starch molecular structure and phosphorylation investigated by a combined chromatographic and chemometric approach. Carbohydr Polym 41: 163–174[CrossRef] Blennow A, Engelsen SB, Nielsen TH, Baunsgaard L, Mikkelsen R (2002) Starch phosphorylation: a new front line in starch research. Trends Plant Sci 7: 445–450[CrossRef][Web of Science][Medline]
Hendriks JHM, Kolbe A, Gibon Y, Stitt M, Geigenberger P (2003) ADP-glucose pyrophosphorylase is activated by post-translational redox-modification in response to light and to sugars in leaves of Arabidopsis and other plant species. Plant Physiol 133: 838–849 Hizukuri S, Tabata S, Nikuni Z (1970) Studies on starch phosphate. Part 1. Estimation of glucose 6-phosphate residues in starch and the presence of other bound phosphate(s). Starch/Staerke 10: 338–343 Hyams J, Davies DR (1972) Induction and characterization of cell-wall mutants of Chlamydomonas reinhardi. Mutat Res 14: 381–389 Kossmann J, Lloyd J (2000) Understanding and influencing starch biochemistry. Crit Rev Plant Sci 19: 171–226[CrossRef] Kuhl A (1962) Zur Physiologie der Speicherung kondensierter anorganischer Phosphate in Chlorella. In Deutsche Botanische Gesellschaft, ed, Beiträge zur Physiologie und Morphologie der Algen. Fischer Verlag, Stuttgart, Germany, pp 157–166 Li CY, Weiss D, Goldschmidt EE (2003) Effects of carbohydrate starvation on gene expression in citrus root. Planta 217: 11–20[Web of Science][Medline] Lorberth R, Ritte G, Willmitzer L, Kossmann J (1998) Inhibition of a starch-granule-bound protein leads to modified starch and repression of cold sweetening. Nat Biotechnol 16: 473–477[CrossRef][Web of Science][Medline]
Mikkelsen R, Baunsgaard L, Blennow A (2004) Functional characterization of
Nielsen TH, Baunsgaard L, Blennow A (2002) Intermediary glucan structures formed during starch granule biosynthesis are enriched in short side chains, a dynamic pulse labeling approach. J Biol Chem 277: 20249–20255 Nielsen TH, Wischmann B, Enevoldsen K, Møller BL (1994) Starch phosphorylation in potato tubers proceeds concurrently with de novo biosynthesis of starch. Plant Physiol 105: 111–117[Abstract]
Reimann R, Ritte G, Steup M, Appenroth KJ (2002) Association of Ritte G, Eckermann N, Haebel S, Lorberth R, Steup M (2000b) Compartmentation of the starch-related R1 protein in higher plants. Starch/Staerke 52: 179–185
Ritte G, Lloyd JR, Eckermann N, Rottmann A, Kossmann J, Steup M (2002) The starch related R1 protein is an Ritte G, Lorberth R, Steup M (2000a) Reversible binding of the starch-related R1 protein to the surface of transitory starch granules. Plant J 21: 387–391[CrossRef][Web of Science][Medline] Ritte G, Steup M, Kossmann J, Lloyd JR (2003) Determination of the starch-phosphorylating enzyme activity in plant extracts. Planta 216: 798–801[Medline]
Shimogawara K, Wykoff DD, Usuda H, Grossman AR (1999) Chlamydomonas reinhardtii mutants abnormal in their responses to phosphorous deprivation. Plant Physiol 120: 685–693
Sueoka N (1960) Mitotic replication of deoxyribonucleic acid in Chlamydomonas reinhardi. Proc Natl Acad Sci USA 46: 83–91 Tabata S, Hizukuri S (1971) Studies on starch phosphate. Part 2. Isolation of glucose-3-phosphate and maltose phosphate by acid hydrolysis of potato starch. Starch/Staerke 23: 267–272
Viksø-Nielsen A, Blennow A, Nielsen TH, Møller BL (1998) Phosphorylated Waffenschmidt S, Jaenicke L (1987) Assay of reducing sugars in the nanomole range with 2,2'-bicinchoninate. Anal Biochem 165: 337–340[CrossRef][Medline]
Wischmann B, Nielsen TH, Møller BL (1999) In vitro biosynthesis of phosphorylated starch in intact potato amyloplasts. Plant Physiol 119: 455–462
Wykoff DD, Davies JP, Melis A, Grossman AR (1998) The regulation of photosynthetic electron transport during nutrient deprivation in Chlamydomonas reinhardtii. Plant Physiol 117: 129–139
Yu T-S, Kofler H, Häusler RE, Hille D, Flügge U-I, Zeeman SC, Smith AM, Kossmann J, Lloyd J, Ritte G, et al (2001) The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter. Plant Cell 13: 1907–1918
Zeeman SC, Tiessen A, Pilling E, Kato KL, Donald AM, Smith AM (2002) Starch synthesis in Arabidopsis. Granule synthesis, composition, and structure. Plant Physiol 129: 516–529 This article has been cited by other articles:
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]+; doubly phosphorylated chains, [DP x (Glc – H2O) + H2O + 3 Na+ + 
]+.
-33P]ATP and 1 µg of GWD for 50 min. Gray and black bars represent the individual measurements of duplicate determinations. The radioactivity present in control samples lacking GWD was subtracted.
