Purification and characterization of non-chloroplastic α-1,4-glucan phosphorylases from leaves of Digitaria eriantha Stent

j. Plant Physiol. \.1JL 149. pp. 501-509 (1996)

Purification and Characterization of Non-Chloroplastic a-1,4-glucan Phosphorylases from Leaves of Digitaria eriantha Stent ROBERT

G.

SHATTERS

Jr. and S. H. WEST

USDA, ARS, Agronomy Dept. University of Florida, Gainesville, FL 32611 Received August 15, 1995 . Accepted January 10, 1996

Summary

We have previously identified four major a-glucan phosphorylase (GP) enzymes in crude leaf extracts from pangolagrass (Digitaria eriantha Stent.). One co-isolates with the chloroplasts (cGP) while the other three are non-chloroplastic (nGP). In this report we present further characterization and leaf cell-type localization of the non-chloroplastic enzymes. In contrast to observations in maize, all of the pangolagrass GP enzymes were present in both isolated bundle sheath strands and mesophyll cells. Ion exchange column chromatography separated the leaf GPs into three peaks: A, Band C. Peak A was the most active and contained three non-chloroplastic GP (nGP), active bands separable by native polyacrylamide gel electrophoresis (NPGE). Peak B GP enzymes migrated identically to peak A enzymes during NPGE. Peak C contained a single chloroplastic GP (cGP). The two major nGPs in the peak A fraction co-purified and migrated as a single band during SOS-PAGE, but they could be separated by IEF-column chromatography. Kinetic properties of these peak A nGP enzymes were similar to those of other plant nGP enzymes, with the exception that the pangolagrass nGP is not inhibited by dinucleotide sugars. The co-localization of the leaf GP enzymes in both bundle sheath and mesophyll cells, and the separation of the non-chloroplastic GPs into two pools (peaks A and B) during ion exchange chromatography are unique characteristics not previously described for plant leaf GPs.

Key words: Digitaria, glucan phosphorylase, pangolagrass, protein purification, carbohydrate metabolism.

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Abbreviations: BSS = bundle sheath strands; cGP = chloroplastic a-l,4-glucan phosphorylase; = dithiothreitol; EOTA = ethylenediaminetetraacetic acid; nGP = non-chloroplastic a-l,4-glucan phosphorylase; GP = a-l,4-glucan phosphorylase; HEPES = N-[2-hydroxyethyl) piperazine-N'-[2-ethanesulfonic acid]; MC = mesophyll cells; NPGE = native polyacrylamide gel electrophoresis; P-5-P = Pyridoxyl-5'phosphate; PMSF = phenylmethylsulfonyl fluoride; PVPP = polyvinylpolypyrrolidone. Introduction

a-l,4-glucan phosphorylase (EC 2.4.1.1), GP, catalyzes the reversible reaction: a-l,4-glucan n +P; ~ a-l,4-glucan n _1 +glucose-l-P. This enzyme has been extensively studied in plants and animals. Although very intricate allosteric and covalent regulatory mechanisms exists to regulate the animal GP activity (Fischer et al., 1970), this level of regulation has not been observed in plants. © 1996 by Gwtav Fischer Verlag, Stuttgart

Plant leaves contain both non-chloroplastic (nGP) (also reported as type I or H glucan phosphorylases) and chloroplastic (cGP) (also reported as type II or L glucan phosphorylases) as first shown by Steup and Latzko (1979). These enzymes have been studied extensively in spinach and pea leaves. Spinach leaves have a single cyrosolic and a single chloroplastic enzyme, whereas in pea there is a single cyrosolie and two chloroplastic GPs (Steup and Latzko, 1979). In both plants the cyrosolic enzyme has a higher apparent affin-

502

ROBERT G. SHATTERS Jr. and S. H. WEST

ity for large branched polyglucans (i.e. starch, amylopectin, and glycogen) and the chloroplastic enzymes are more active with smaller oligoglucans (with degrees of polymerization greater than 4). Because of the low affinity of cGP for high molecular-mass glucans and the inability of purified cGP to degrade starch granules in vitro (Steup et al., 1983), it is assumed that transitory starch granules within the chloroplast are first acted upon by endoamylases (Steup and Schachtele, 1981, and Steup, 1988). The soluble glucans produced would then be degraded to glucose-l-P and small oligoglucans by the action of cGP' Near complete conversion of the glucans to hexose sugars could be aided by the action of D-enzymes (4-a-Glucanotransferase) and other transglycosidase enzymes that link small oligoglucans to produce larger polymers that are better substrates for the cGP (for review of starch degrading enzymes see Stitt and Steup, 1985 and Steup, 1990). There is currently no working hypothesis for the function of the non-chloroplastic Gp, especially since this enzyme utilizes large branched polysaccharides as preferential substrates. Yang and Steup (1990) have isolated a high molecular-mass cytosolic heteroglycan from spinach and pea leaves that interacts strongly with the nGP' However, the function, structure, or importance of this polysaccharide is unknown. Compartmentation of leaf GP activity has been studied in maize, a C 4 plant (Mateyka and Schnarrenberger, 1984 and Echeverria and Boyer, 1986). Both nGP and cGP enzymes were observed in maize, but expression of these enzymes was cell specific. The chloroplastic enzyme was limited to the bundle sheath strands (BSS) while the non-chloroplastic enzyme was only found in the mesophyll cells. Bundle-sheath cell-specific localization of the cGP enzyme was explained by correlation with BSS specific build-up of transitory starch in maize leaves under standard light conditions (Woo et al., 1971; Downton and Hawker, 1973). We are studying the carbohydrate metabolism in the leaves of pangolagrass (reclassified from Digitaria decumbens to Digitaria eriantha) a warm-season C4 grass species. Hilliard and West (1970) showed that this grass accumulated significant amounts of transient starch in both the mesophyll and bundle-sheath cells under normal greenhouse diurnal conditions. Because this differs from what has been observed in maize, we have hypothesized that cell-type-specific-GP compartmentation in D. enantha may be different than that observed for maize. In experiments designed to test this hypothesis, we have characterized the cell-specific expression ofleaf G Ps in D. eriantha and purified the n G P enzymes.

Materials and Methods

Plant material 1 Pangolagrass (Digitaria eriantha Stent.) plants were propagated clonally from stolons and grown in 15 em pors in metro mix 350 1 Names of vendors are included for the benefit of the reader and do not imply endorsement or preferential treatment by the United States Department of Agriculture.

potting soil (Grace/Sierra Horticultural Products Co., Milpitas, CAl in a greenhouse, watered daily and fertilized weekly with 4.5 g of Peters 20-20-20 (Grace/Sierra Horticultural Products Co., Milpitas, CA) and 1.5 g of Minor L nutrients (Voluntary Purchasing Groups, Inc., Bonham, TX) per pot. Greenhouse temperatures ranged between 25·C and 33 ·C during the days and 30·C and 24 ·C during the nights, under natural diurnal light cycles. Plants established in pots for approximately two months were cut back to a 10cm stubble and allowed to regrow for three weeks before harvesting. Leaves used for cell separation and enzyme purification were removed immediately following the night-time incubation.

Separation ofleaf bundle sheath and mesophyll cells Bundle sheath strands (BSS) and mesophyll cells (MC) were separated using slight modifications of the procedure described by Edwards and Black (1971). Twelve grams of the first and second fully expanded leaves were removed from plants incubated over-night in the dark in a growth chamber at 25 ·C and cut with scissors into 1-3 mm size ~ieces. These were ground in 72 mL of LCS buffer (0.33 mol· L- sorbitol; 50 mmol . L-I Tricine-NaOH, pH S.O; 2 mmol . L-I NaN0 3; 2 mmol . L-I EOTA; 1 mmol . L-I MnCl 1 ; 5 mmol· L-I MgCb and 5 mmol. L-I K1HP0 4) with a mortar and pestle on ice for 10 min. An additional 72 mL of LCS buffer were added and the homogenate was filtered through a tea strainer. The unmacerated tissue was returned to the mortar. The filtrate was sequentially passed through 500, SO, 40 and 30 ~m nylon mesh filters and the retentate from each was combined with the unmacerated tissue. The filtrate from the 30 ~m filtration contained primarily mesophyll cells and was then passed rhrough a 20 ~m mesh. Mesophyll cells were retained on this mesh and were rinsed 3 times with 20 mL of LCS buffer and then collected in 20 mL of LCS buffer. These mesophyll cells were centrifuged at 1,000 Kn for 1 min and the supernatant was removed. The cell pellet was resuspended in 5 mL of GP buffer (25 mmol· L-I HEPES, pH 7.5; 5 mmol. L-I MgCll; 0.5 mmol· L-I EOTA; 3 mmol· L-f DTT; 1 mmol L-I PMSF; 0.01 % (w/v) BHT; 0.6 % (w/v) PVPP; and 10 % (v/v) glycerol) and stored at -SO·C until used. Purity of cells was checked by microscopic analysis and coomassie staining of SOS-PAGE-separated proteins from crude extracts of each cell type. Bundle-sheath strands were prepared from the unmacerated tissue by adding 30 mL of LCS buffer and gently grinding for an additional 5 min. Sixty more milliliters of LCS buffer were added, and the sample was passed through a 500 ~m mesh filter and then an SO ~m mesh filter. Cells retained on the SO ~m mesh filter were rinsed 3 times with 20 mL of LCS buffer and collected in approximately 20 mL of LCS buffer. The cell suspension was added to a 15 mL Ten-Broek tissue grinder and ground for three strokes. The solution was again passed through an so ~m mesh filter, and the retained cells were washed 3 times with 20 mL of LCS buffer, collected in 10 mL of LCS buffer, and centrifuged at 1,000 Kn for 1 min. The supernatant was removed, and the bundle sheath cells were resuspended in 5 mL of GP buffer. All cell samples were frozen at -SO·C until used. Before using for enzyme assays, the frozen BSS and MC were broken by grinding in liquid nitrogen to a fine powder in the presence of a protease inhibitor cocktail (200 X stock contains 20 mmol . L-I EDTA, 20 mmol· L-I Mg(OAch, 2000 unirs/mL Trasylol, 0.2 mmol· L-I antipain, 0.2 mmol · L-I leupeptin, 0.2 mM pepstatin) (YaOeau and Blobel, 19S9). The powder was transferred to microcentrifuge tubes and centrifuged at 10,000 Kn for 10 min at 4°C. The supernatant was used directly for enzyme assays and gel electrophoresis experiments.

Leaf a-glucan phosphorylases from Digitaria eriantha

Glucan phosphorylase purification Typically, 100 g of Pangolagrass leaves were ground to a fine powder in liquid nitrogen with a mortar and pestle. The powder was transferred to a blender with 500 mL of GP buffer and blended on high for 10 s. Blending was repeated 5 times. The homogenate was then filtered through a combination of 4 layers of cheesecloth and one layer of miracloth. The filtrate was centrifuged at 25,000 & for 30 min and the supernatant was used directly for NH 4(S04h precipitation. The majority of the GP activity was precipitated between 37% and 70 % NH4 (S04h. The 70 % NH4 (S04h pellet containing the majority of the GP activity was resuspended in approximately 50 mL of GP buffer and dialyzed against 4 L of GP buffer with 2 changes. The dialyzed sample was again centrifuged, and the supernatant was directly loaded onto an HR 16/ 10 MonoQ column using a Pharmacia FPLCm system. Activity of a-1,4-glucan phosphorylase was eluted in a 0 to 0.5 mol· L-1 KCi gradient. Alpha-1,4glucan phosphorylase fractions contained within the major GP-active peak were used for further purification steps. Two separate procedures were used in the subsequent purification schemes. Procedure I: A starch-sepharose column was synthesized by bonding soluble starch to an epoxy-activated sepharose 6B material using the protocol provided by the supplier (Pharmacia, Piscataway, NJ). This column was used as an affinity column for GP purification as described by Steup (1990). Alpha-1,4-glucan phosphorylase eluted as a single protein band when analyzed by SDS-PAGE. Purifcation II: The MonoQ peak A fraction was concentrated to 5 mL using Centripreptm concentrators (Amicon, Beverly, MA) and loaded onto a Highload 16/60 Superdex 200 size exclusion column (Pharmacia). Fractions containing the GP active peak were pooled, and the GP was finally purified by isoelectric focusing on a Pharmacia HR 5120 mono P column through a pH gradient of 4.0 to 7.0. Fractions containing GP activity were pooled, and used for further analysis.

Glucan phosphorylase assay A previously described a-1,4-glucan phosphorylase specific assay (Shatters, et aI., 1995) was used to determine GP activity in chromatography fractions. This assay was down-sized to a 200 ilL volume and performed in micro titer plates using a Bio-TeK EL340I microplate reader (Bio-Tek Instruments, Inc., Burlington, VT). The assay coupled the GP-mediated production of Glucose-I-Phosphate from amylopectin to NADPH production through the combined activities of phosphoglucomutase and glucose-1,6-phosphate dehydrogenase, which was monitored spectrophotometrically at A340. An alternative two-step assay was used in experiments to determine the pH and temperature optima for the purified GP. In the first step, a-1,4-glucan phosphorylase was allowed to act on the glucan substrate for 30 min without the presence of phosphoglucomutase or glucose-1,6-phosphate dehydrogenase. Reactions were stopped by boiling for 1 min. Phosphoglucomutase, glucose-1,6-phosphate dehydrogenase, and NADP were then added, and NADPH production was monitored spectrophotometrically at A340'

Gel electrophoresis Native polyacrylamide gel electrophoresis (NPGE) and the GP activity staining was performed as described by Shatters et aI. (1995). Enzymes with a-1,4-glucan phosphorylase activity were detected using a starch synthesis method with glycogen as the primer, and KI/Iz staining of the synthesized starch (Steup and Latzko, 1979). Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was performed following the procedure of Fling and Gregerson (1986).

503

Results

Bundle sheath and mesophyll cell expression of GP Bundle sheath and mesophyll cells were successfully separated using the described sieving method (Fig. 1). Only minor contamination of one cell type with the other was observed by microscopic observation of the BSS and MC fractions, with slightly more appearance of MC in the BSS fraction than vice versa (Fig. 1 A). Actual counting of cells in the different fractions indicated less than 10 % contamination of each cell type with the other (data not shown). Also, distinct protein banding patterns were observed when proteins in crude extracts from each were separated by SOS-PAGE and stained with Coomassie blue (Fig. 1 B). Only a faint band was visible in the BSS lane at the position equal to that of the darkest staining band in the MC extract lane and vice versa. The same four GP bands were detected in both cell types when extracts from each were used for NPGE, and GP activity was detected by staining for glucose-1-phosphate-dependent starch synthesis (Fig. 1 C). The top three bands corresponded to the nGP enzymes, and the fastest migrating band corresponded to the cGP enzyme (Shatters et al., 1995).

MonoQ.fractionation ofpangolagrass leafGP activity Ammonium-sulphate-precipitated GP enzymes from leaf crude extracts were separated into three peaks when chromatographed on an FPLC MonoQ column (Fig. 2). These three peaks, A, B, and C, eluted at 80, 114, and 260 m mol. L-\ KCl, respectively (Fig. 2). Proteins in each peak were separated by NPGE and visualized by staining for G-1-P-dependent starch synthesis (Fig. 2, inset). Peaks A and B contained three GP-active bands corresponding to the nGP enzymes (Shatters and West, 1995). The migration of these three bands through the gel was identical for both peaks A and B. Peak C contained a single band that migrated more rapidly during NPGE than either peak A or B enzymes. This band corresponded to the chloroplast enzyme previously identified (Shatters and West, 1995).

Purification ofpeak A nGP using procedure I The pangolagrass nGP peak A enzymes were purified by starch-Sepharose column chromatography by eluting the nGP from newly synthesized starch Sepharose columns with a to 5 % dextrin gradient (Fig. 3 A). The purified nGP enzymes were analyzed both by activity staining of NPGE separated proteins and by total protein staining of SOS-PAGE separated peptides (Fig. 4). Although SOS-PAGE analysis detected a single peptide with a Mr of 94 ku (kDa), NPGE separated the active G-1-P-dependent starch biosynthetic activity as a doublet corresponding to the major nGP enzymes in crude extracts. When a starch-sepharose column was used for a secondtime, a different elution pattern for GP activity was observed (Fig. 3 B). During this second use of the column, the majority of the GP activity now eluted with a short retention time and before the application of the dextrin gradient. No further activity eluted during the application of the dextrin gradient

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Purification ofpeak A nGP activity using procedure II The GP activity eluted from a Highload 16/60 Superdex 200 size-exclusion column at a time consistent with a protein with a molecular mass of 200 ku (Fig. 5 A). This is approximately twice the size of the single band observed by SOSPAGE of the GP purified using procedure I, suggesting that the active nGP exists as a dimer in vivo. This finding is in agreement with all other observations about dicot leaf GP structure (Steup, 1990). The GP active peak eluted from the superdex column was applied directly to a MonoP column and was eluted under isoelectric focusing conditions with a pH range between 4.0 and 8.0 (Fig. 5B). The GP activity eluted as two major peaks,

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Characterization ofnGP activity The pangolagrass enzyme(s) were most active at higher temperatures and remained active up to 55 ·C, the highest temperature used in the experiment (Fig. 6). High enzyme activity at this elevated temperature was also shown for one GP enzyme purified from banana fruit (Singh and Sanwal, 1976). The pH optimum for the pangolagrass leaf nGPs was between 5.0 and 6.0 using amylopectin as the substrate (Fig. 6). This optimal pH range was slightly more acidic than that

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observed with spinach GP activity, which had an optimal pH range of 6.0 to 6.5 (Shimomura et al., 1982). The nGP enzymes had a substrate preference for large molecular mass and branched glucans, with the order of substrate preference from best substrate to worst being: amylopectin, amylose, glycogen, maltoheptaose, and maltohexaose (Fig. 7). This substrate preference is similar to that for the well characterized dicot nGP enzymes (Steup, 1990). Potential enzymatic inhibitors were also used in experiments to determine their effect on pangolagrass nGP (Table 1). Although at concentrations below 2 m mol· L-1, both UPO-glucose and

Fig. 4: Electrophoretic characterization of purified nGP. A. SDSPAGE separation and silver staining of total proteins from the different GP purification steps. Numbers on left border represent location of molecular mass markers, lanes 1 and 2, molecular mass markers; lane 3 leaf crude extract; lane 4, NH4(S04h precipitate; lane 5, Mono Q peak A (Fig. 1); lane 6, starch-Sepharose first-run elution of GP activity with 0-5 % dextrin gradient (Fig. 2A); lane 7, starch-Sepharose second-run GP activity eluted without a dextrin gradient (Fig. 2 B). B, NPGE separation and starch biosynthetic staining of GP activity from first run elution of starch sepharose purified nGP, Lanes 1-3; 20, 40, 60Jlg ofGP purified from first use of the starch sepharose column, respectively; lanes 4 and 5; 40 and 60 Jlg of GP purified from the second use of the starch sepharose column, respectively.

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Discussion

D. eriantha, a C 4 warm-season grass species, which was previously shown to synthesize large amounts of transitory starch in both the bundle sheath and mesophyll cells (Hil-

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pH Fig. 6: Physical parameters of nG P activity purified using procedure I. A, Effect of temperature on amylopectin degrading activity of purified nGP enzymes. B, Effect of pH on amylo~ectin degrading activity of purified nGP enzymes. P, 50 mmol · L - NaP0 4 buffer; M, 50 mmol · L -1 MOPS buffer; and H, 50 mmol· L -1 HEPES buffer.

liard and West, 1970), expresses all of the leaf nGP and cGP enzymes in both leaf cell types. In comparison, previous work in maize has shown that the nGP enzyme was present in the MCs, while the BSS contained the cGP enzyme (Mateyka and Schnarrenberger, 1984). This cell-type specific expression was interpreted as being a C4-plant-specific modification, associated with the differences in the carbohydrate metabolism that occur between the separate cell types. Under normal greenhouse conditions, maize leaves synthesize most of the transient starch in the BSS with very little starch deposition in the chloroplasts of the MCs (Downton and Hawker, 1973). If leaf cGP is involved in starch degradation, it is then understandable why no cGP activity was detected in MCs. D. eriantha leaves synthesize large quantities of transient

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Table 1: Effect of potential a-l,4-glucan posphorylase inhibitors on starch biosynthetic activity of pangolagrass purified non-chloroplastic a-l,4-glucan phosphorylase.

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56

* All reactions were performed with amylopectin as the substrate. Control activity was defined as the tOtal activity measured in identical reactions but with no potential inhibitors. starch in both the BSS and the MCs under standard greenhouse conditions (Hilliard and West, 1971). Therefore, we hypothesized that if cell-specific expression of the cGP is linked to starch metabolism, it should be expressed in both cell types. Our results support this hypothesis. It was interesting to find that the nGP enzymes were expressed in both the MCs and the BSS. Although the function of the non-chloroplastic GP enzyme is still unknown, further analysis of the differential cell-specific expression between maize and pangolagrass may provide more information on the role and regulation of these enzymes. Our work shows that pangolagrass leaves contain at least two major non-chloroplastic a-l,4-glucan phosphorylase enzymes. This is the first report of multiple non-chloroplastic GP enzymes in plant leaf tissue. One GP form is apparently not the result of proteolytic modification of the other, since they both migrate as a single band during 50S-PAGE. However, the separation of these two forms during NPGE and IEF column chromatography indicates charge differences between the nGPs. Digitaria eriantha (formerly named Digita-

ria decumbens) is a sterile triploid (Oakes, 1969), and it is therefore possible to explain the multiple nGP bands as allelic variations between comparable nGP loci from the ancestral parents of pangolagrass. However, since we observed not only multiple nGP bands by NPGE but also separation of each of these bands into two groups based on elution from a monoQ ion exchange column (peaks A and B, Fig. 2), the interpretations of these results must be more complicated. Using OEAE-cellulose column chromatography, two peaks of GP activity were identified in crude extracts from maize leaves: one corresponding to the nGP activity, and the other to cGP activity (Mateyka and Schnarrenberger, 1984). In pangolagrass the nGP activity is resolved into two peaks by MonoQ FPLC ion exchange column chromatography. Both of these fractions contain nGP activity that migrates identically during NPGE. It will be important to determine the nature of the difference between the GP enzymes separated in peaks A and B since no protein modifications have been described for plant GP's despite intricate covalent regulation of mammalian Gp. This is also the first report of nGPs in monocot leaves that are similar to dicot leaf nGPs. Alpha-l,4-glucan phosphorylase has been purified and studied from different monocot tissues including banana leaves (Kumar and Sanwal, 1982 and 1988), banana fruit (Singh and Sanwal, 1976), and maize kernels (Tsai and Nelson, 1968, 1969; Lee and Braun, 1973; and Burr and Nelson, 1975). The enzyme from banana leaves was identified as a homotetramer with a subunit molecular mass of 55 ku, which is quite different from the GP enzyme described in dicot leaves and the pangolagrass nGP enzymes described in this paper. The pangolagrass nGPs with a native molecular mass of approx. 200 ku are similar in size to the major GP identified in maize kernels, with an estimated molecular mass of 223,000 ± 10,000 ku (Burr and Nelson, 1975). This size and dimer configuration is similar to that shown for the spinach nGP enzymes (Steup et al., 1980; and Steup, 1981). The enzymatic characteristics of the pangolagrass leaf nGP enzymes are quite similar to those of the dicot leaf nGPs (Steup and Schachtele, 1981; and Shimomura et al., 1982), with the exception that pangolagrass nGP activity was not inhibited by UOP- or ADP-glucose. Therefore, this class of enzymes must have a similar role in the metabolism of nonchloroplastic glycans in leaves of both C 3 and C4 plants. In the case of pangolagrass, these putative non-chloroplastic glycans must exist in both the bundle sheath and mesophyll cells, whereas, in maize they are primarily in the MCs. We hope that by further studying the properties and regulation of these enzymes, we will be able to identify their role and importance in leaf carbohydrate metabolism. Acknowledgements

The technical assistance of Janet Sasser is gratefully acknowledged. We also thank Dr. Richard Wheeler for insightful discussions. References

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