Plant Physiol. (1987) 85, 1008-10150032-0889/87/85/1008/08/$0 1.00/0

Inhibition of Mung Bean UDP-Glucose: (1-m3)-fi-GlucanSynthase by UDP-PyridoxalEVIDENCE FOR AN ACTIVE-SITE AMINO GROUP

Received for publication February 24, 1987 and in revised form August 11, 1987

STEVE M. READ*' AND DEBORAH P. DELMER2ARCO Plant Cell Research Institute, Dublin, California

ABSTRACT

UDP-pyridoxal competitively inhibits the Ca2`-, cellobiose-activated(1-.3)-#-glucan synthase activity of unfractionated mung bean (Vignaradiata) membranes, with a K, of 3.8 1 0.7 micromolar, when addedsimultaneously with the substrate UDP-glucose in brief (3 minute) as-says. Preincubation of membranes with UDP-pyridoxal and no UDP-glucose, however, causes progressive reduction of the V,., of subse-quently assayed enzyme and, after equilibrium is reached, 50% inhibitionoccurs with 0.84 ± 0.05 micromolar UDP-pyridoxal. This progressiveinhibition is reversible provided that the UDP-pyridoxylated membranesare not treated with borohydride, indicating formation of a Schiff's basebetween the inhibitor and an enzyme amino group. Consistent with this,UDP-pyridoxine is not an inhibitor. The reaction of (I-.3)-t,-glucansynthase with UDP-pyridoxal is stimulated strongly by Ca2 and, lesseffectively, by cellobiose or sucrose, and the enzyme is protected againstUDP-pyridoxal by UDP-glucose or by other competitive inhibitors,implying that modification is occurring at the active site. Pyridoxalphosphate is a less potent and less specific inhibitor. Latent (1-.3)-#-glucan synthase activity inside membrane vesicles can be unmasked andrendered sensitive to UDP-pyridoxal by the addition of digitonin. Treat-ment of membrane proteins with UDP4[3Hlpyridoxal and borohydridelabels a number of polypeptides but labeling of none of these specificallyrequires Ca2' and sucrose; however, a polypeptide of molecular weight42,000 is labeled by UDP4-3Hjpyridoxal in the presence of Mg2" and co-purifies with (1-_3)-O-glucan synthase activity.

All living plant cells appear to make the (l1--3)-fl-glucan calloseat their plasma membrane primarily as a wound-response,whether the wound is mechanical, due to assault by a pathogen,or due to various other kinds of stress (1, 10, 12, and referencestherein). In addition, callose is found at particular times in thedifferentiation of certain unwounded cells, such as cotton fibers,pollen tubes, sieve plates, and the cell plate (5). Successful invitro assay of (1--3)-fl-glucan synthase at rates approachingphysiological polymerization rates requires that the enzyme beactivated by micromolar levels of Ca2l and by a ,B-glucoside orsucrose (8, 12, 19, 29). These effectors act synergistically, andthe enzyme then displays Michaelis-Menten kinetics and has aKm for its substrate UDP-glucose of 0.2 to 0.3 mm (8, 29).Polyamines (13) or Mg2e (8) can stimulate the enzyme when

Current address: Plant Cell Biology Research Centre, School ofBotany, University of Melbourne, Parkville, Victoria 3052. Australia.

2Current address: Department of Botany, Institute of Life Sciences,The Hebrew University, Jerusalem, Israel.

Ca2" levels are not saturating. Digitonin stimulates (1- 3)-fl-glucan synthase by up to several-fold, and this detergent increasesthe Vmax of ( l->3)-,3-glucan synthase with no effect on its Km forUDP-glucose (8). The enzyme, previously called Glucan Syn-thase II, is located in the plasma membrane (23), and is of largesize but to date has only been partially purified (6, 8).At the molecular level, little is known about any ofthe enzymes

that catalyze the synthesis of polysaccharides in higher plants.We therefore decided to attempt to identify the catalytic poly-peptide of the (1--3)-f3-glucan synthase enzyme protein in un-fractionated membranes by a chemical modification approach.Pyridoxal-P has frequently been used as a modifying reagent forlysine residues at the active sites of purified enzymes (1 1, 15, andreferences therein), and reduction of the resultant Schiffrs baseswith borohydride gives stable e-N-pyridoxyl-lysine residues. Ad-dition of a nucleoside moiety can direct pyridoxal -P to nucleo-tide-binding sites (27, 28); thus, UDP-pyridoxal selectively mod-ified an active-site lysine residue of purified rabbit muscle gly-cogen synthase, producing an E-N-(UDP)pyridoxyl-lysine residue(27). Specific radiolabeling of purified proteins has been success-fully performed using this reaction with the isotope originatingeither as NaB3H4 (20) or as [3H]pyridoxal-P (15, 21) or ADP-[3HJpyridoxal (28). Here we describe the effect ofUDP-pyridoxalon the (1--3)-fl-glucan synthase activity of mung bean mem-branes and show the polypeptides labelled by UDP-[3H]pyridoxalunder various conditions. We also use UDP-pyridoxylation toshow that stimulation by digitonin of membrane-bound (1- 3)-,3-glucan synthase activity occurs by permeabilization of vesiclesto substrate UDP-glucose by the detergent. A preliminary reportof some of this work has already appeared (25).

MATERIALS AND METHODS

Materials. Mung bean (Vigna radiata cv Berkin) seeds wereobtained from ARCO Seed Co., El Centro, CA, and were soakedovernight in aerated water before being sown in vermiculite andgrown for 3 to 5 d at 27°C in complete darkness. UDP-[U-'4C]glucose (240 mCi/mmol) was from I.C.N., NaB3H4 (10.3 Ci/mmol) was from N.E.N., digitonin was from Serva, activatedMnO2 was from Aldrich, Aquamix was from Westchem, andUDP, UDP-xylose, unlabeled UDP-glucose, leupeptin, andPMSF3 were from Sigma. Sephadex G-10 was from Pharmacia,DE-52 was from Whatman, and Dowex AG IX-8, 100 to 200mesh, was from Bio-Rad. All other chemicals were ofthe highestpurity available.UDP-pyridoxal was obtained from Dr. T. Fukui, Osaka Uni-

3Abbreviations: PMSF, phenylmethylsulfonyl fluoride; FPLC, fastprotein liquid chromatography; Ki., concentration ofinhibitor for half-maximal initial rate of inhibition.

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INHIBITION OF (1--3)-fl-GLUCAN SYNTHASE BY UDP-PYRIDOXAL

versity, Japan, and was stored as a 4 mm stock solution at -20°Cin 100 mm Hepes/KOH, 1 mM EDTA (pH 7.8). Pyridoxal-P(Sigma) was purified by anion-exchange chromatography on a1.0 x 14 cm column of AG 1-X8, acetate form, eluted with agradient of increasing concentration of acetic acid (26). Thepyridoxal-P peak, detected by its A388 in alkali, was eluted at 4.0to 4.5 M acetic acid, and was diluted to about 1.0 M acid,lyophilized, redissolved in water, relyophilized, and stored at-20°C. Stock solutions were adjusted to pH 7.8 with KOH. Allpyridoxal derivatives and modified proteins were handled underdim lighting wherever possible.

Preparation of Membrane Proteins. Hypocotyls and roots ofetiolated mung bean seedlings were homogenized in an equalweight of 50 mm Hepes/KOH, 5 mM EDTA, 1 mM EGTA (pH7.3) containing 0.5 mm PMSF, 0.05 mm leupeptin, for 5 s in aWaring Blendor, followed by 1 pass with a Teflon homogenizer.Sorbitol (250 mM) was included on occasion as indicated. Themixture was filtered through four sheets of cheesecloth, centri-fuged for 5 min in a Sorvall SS34 rotor at 2500g, the pelletdiscarded, and the supematant centrifuged for 30 min in aBeckman 7OTi rotor at 75,000g. This membrane pellet waswashed once by resuspension in 50 mM Hepes/KOH, 1 mMEDTA (pH 7.3) containing 250 mm sorbitol where appropriate,recentrifuged for 30 min at 75,000g, and finally resuspended inthis buffer to one-fifth of the volume of the original homogenateand stored at -80°C. The yield of membranes ranged from 0.5to 1.0 mg protein/g tissue, and was determined by Coomassieblue G dye-binding (24).Membrane proteins were solubilized in 50 mM Hepes/KOH

(pH 7.3), 20% glycerol, containing 1% digitonin, as described(8). (1--3)-f,-Glucan synthase was partially purified by glycerolgradient centrifugation in the presence of 1 mM EDTA (8),followed by anion-exchange chromatography on a PharmaciaMono-Q FPLC column equilibrated in 10 mM Tris/HCI (pH7.5), 20% glycerol, 0.03% digitonin. Protein was eluted with alinear gradient of NaCl in this buffer, and exchanged into 50 mMHepes/KOH (pH 7.8), 20% glycerol, by ultrafiltration in anAmicon pressure cell.Assay of (1-+3)-j-G1ucan Synthase. Assays were performed in

triplicate at 25°C in 200 Al volumes of 50 mM Hepes/KOH, 5mM MgCl2, 5 mm CaCl2 (pH 7.0), containing 10 mM cellobioseor 100 mm sucrose, 0.01 to 0.03% digitonin, 100 AM to 1.0 mMUDP-['4C]glucose (200,000 to 400,000 cpm/assay) and 10 to 24,ug of protein, unless otherwise stated. Reactions were terminatedafter 3 or 10 min by addition of 3 ml ice-cold 66% ethanolcontaining 0.5 mM EDTA, carrier cellulose was then added, andthe mixtures chilled at -20°C for 30 min and then filtered ontoWhatman GF/C glass-fiber filters, which were washed three timesin 66% ethanol, once in chloroform:methanol 1:1, and countedin Aquamix. Typical activities for unfractionated membraneswere 60 nmol glucose/min-mg protein at 1 mM UDP-glucose,with a Km for UDP-glucose of 0.25 mm. The radioactive productunder these conditions is essentially all linear (1-*3)-,B-glucan(8).

Reaction with Inhibitors. The ability of pyridoxal derivativesto act as competitive inhibitors of (l- 3)-j3-glucan synthase wasmeasured by addition of the inhibitor simultaneously with vary-ing concentrations of UDP-['4C]glucose to start the assay. Theassay duration was 3 min.

Reaction of inhibitors with membranes before assay was gen-erally monitored by preincubation in 80 Al volumes at OC undervarious conditions, before removal of three 20 Al aliquots for 10-fold dilution into assay cocktail (see above) containing 1 mmUDP-['4C]glucose and assay at 25°C for 3 min. On occasion,UDP-pyridoxal was allowed to react with membranes at OC involumes of up to 180 AI before addition of effectors as necessary

for 3 min. Activities reduced by preincubation with inhibitorswere always compared with control tubes in which the corre-

sponding final concentration of inhibitor was added togetherwith the UDP-[ 14C]glucose at the start of the assay. Initial ratesof reaction were determined by removing samples for assay at20 s intervals after addition of UDP-pyridoxal.

Kinetic constants Km and V. were determined by the DirectLinear technique (4), and Ki values were determined from plotsof apparent Km values versus inhibitor concentration or fromDixon plots of l/v versus inhibitor concentration. To measurethe overall equilibrium constant for Schiffis base formationbetween (1--3)-f3-glucan synthase and UDP-pyridoxal, mem-branes were incubated with the inhibitor for 30 to 60 min beforeassay as above; since the enzyme activity then remaining wasconstant over time and dependent solely on the UDP-pyridoxalconcentration, it was assumed that negligible breakdown of theinhibitor was occurring. The equilibrium constant was deter-mined from a plot of % inhibition/% residual activity againstUDP-pyridoxal concentration, which was linear with a slope ofl/KY. Straight lines were fitted by unconstrained least-squaresanalysis.

Synthesis of UDP-13HjPyridoxal. UDP-[3H]pyridoxal was syn-thesized from unlabeled UDP-pyridoxal by a modification ofthemethod of Stock et al. (26), with the UDP-[3H]pyridoxal beingreoxidized under milder conditions. UDP-pyridoxal (100 ,l of a4 mm solution in 100 mM Hepes/KOH, 1 mM EDTA, pH 7.8)was mixed at 0°C with 100 ,l 10 mM NaOH containing 2.46 mMNaB3H4 (2.53 mCi). After 15 min at 0°C, 10 Ml 0.3 M unlabeledNaBH4 in 10 mM NaOH was added, and after a further 15 minthe pH was adjusted to 7.0 with 1.0 M Hepes (acid form) and thevial warmed to 20°C and allowed to stand for 2 h. ActivatedMnO2 (2.6 mg) was then added and the mixture stirred at 20°Cfor 3 h. The progress of reoxidation was followed spectroscopi-cally by removing 10 gl of the mixture, adding 590 ul 0.1 M

NaOH, centrifuging for 1 min at 15,000g in an EppendorfMinifuge to remove solid MnO2, and measuring the A of thesupematant between 250 and 400 nm against a similar super-natant from a mix containing only buffer and MnO2; after 3 hthe A388 measured in this way had risen from 0.009 to 0.137,indicating about 60% reoxidation. The MnO2 was then removedby centrifugation at 1 5,000g for 5 min.The brown-yellow supematant and washings were loaded on

to a 1.0 x 15 cm column of DE-52 equilibrated in 1 mmtriethanolamine-HCl (pH 7.0), 10 mM LiCl, which was washedwith 60 ml of this buffer until the 3H20 background was minimal,and then eluted with 10 mm HCI, 10 mm LiCl. The radioactivepeak eluting with the acid front contained both UDP-[3H]pyri-doxal and UDP-[3H]pyridoxine as judged spectroscopically andfrom TLC analysis (see below), and was lyophilized, dissolved in1.0 ml water, and desalted on a 0.8 x 20 cm column of SephadexG-10 equilibrated in 7.5 mM Li acetate (pH 5.0). The samplewas then rechromatographed on the DE-52 column equilibratedin this buffer, and eluted with a 160 ml gradient of 7.5 to 300mM Li acetate (pH 5.0). UDP-[3H]pyridoxine (A maximum at308 nm) eluted after 137 ml, and UDP-[3H]pyridoxal (A maxi-mum at 388 nm in 0.1 M NaOH) eluted at the end of thegradient. The UDP-[3H]pyridoxal fractions were lyophilized,redissolved in water, and gel-filtered on Sephadex G-10 into 5mM Hepes (pH 7.8), 1 mM EDTA, and stored at -80°C. Yield,16%; specific activity, 0.78 Ci/mmol.The radiochemical purity of fractions was analyzed by TLC

on Merck 60 F-254 silica gel plates developed in n-butanol:aceticacid:water 12:6:9 by volume, using approximately 10 nmol ofunlabeled sample as carrier. After air-drying, the position ofunlabeled compounds was determined by viewing in short-wave-length ultraviolet light, and the distribution of radioactivity was

and 20 Jd 10 mM UDP-['4C]glucose, followed by assay at 25°C

1009

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Plant Physiol. Vol. 85, 1987

fluorography on Kodak X-Omat AR film at -80°C, or byscraping areas of the gel into 0.5 ml water, shaking for 30 min,then adding 3.0 ml Aquamix and scintillation counting. The RFof UDP-pyridoxal in the above solvent was 0.28, and UDP-[3H]pyridoxal gave a single radioactive spot that ran exactly with theunlabeled material and contained 92% of the recovered radio-activity. The RF of UDP-pyridoxine, produced by reduction ofUDP-pyridoxal with NaBH4, was 0.16, and the RFs ofthe reducedSchiff's bases, formed by treatment of UDP-pyridoxal with anexcess of Tris, serine, or N-a-acetyl-lysine at pH 10 followed byreduction with NaBH4, were 0.13, 0.10, and 0.115, respectively;in every case, the reaction products from UDP-[3H]pyridoxalgave single radioactive spots, containing over 91% of the re-

covered radioactivity, that migrated with the unlabeled standards(not shown).

Reaction of Proteins with UDP_[3HjPyridoxal. Mung beanmembrane proteins, digitonin-solubilized proteins, and partiallypurified preparations of(l--.3)-p-glucan synthase were treated atOC with UDP-[3H]pyridoxal in 50 mm Hepes/KOH (pH 7.8),in the presence of various effectors or inhibitors, then reducedwith NaBH4. Control samples were treated with UDP-[3H]pyri-doxine instead of UDP-[3H]pyridoxal, or reacted with cysteam-ine instead ofbeing reduced with NaBH4. Aliquots were removedfor assay of (1--3)-#-glucan synthase activity (see above), andthe remainder mixed with an equal volume of ice-cold 25%TCA. After standing for 15 min at OC, the precipitated proteinswere pelleted by centrifugation for 15 min at 15,000g, washedonce in ice-cold 12.5% TCA, then redissolved in 10

A SDS-sample buffer (0.125 M Tris/HCl [pH 6.8], 4 M urea, 20%glycerol, 3% SDS, 0.1 M DTT, 1 mM EDTA, 0.006% bromophe-nol blue) and heated at100°C for1 min. Aliquots were removedfor scintillation counting and the remainder analysed by SDS-PAGE on 0.75 mm thick mini-gels, using the buffers of Laemmli(16). Gels were stained with Coomassie blue R, or impregnatedwith Amplify (Amersham) and dried for fluorography on pre-flashed (17) Kodak X-Omat AR film at -80°C. ['4C]Methylatedmarker proteins were obtained from Amersham. Densitometrywas performed using a Helena Quick Scan R&D densitometer.

RESULTS

Inhibition of (1l--3)-ft-Glucan Synthase by UDP-Pyridoxal.When UDP-pyridoxal was added simultaneously with UDP-['4C]glucose at the start of the assay period, a reduction in Ca2l-,cellobiose-activated (I-.3)-j#-glucan synthase activity was ob-served. If the assay was of short (3 min) duration, inhibitionfollowed competitive kinetics (not shown), and the Ki for UDP-pyridoxal was measured as 3.8 ± 0.7AM (4 determinations) atpH 7.8, implying a high affinity for the enzyme.

Progressive inhibition, however, with a reduction in V,,,occurred on preincubation of membranes with UDP-pyridoxalat0°C in the presence of enzyme activators before dilution intobuffer containing UDP-['4C]glucose and assay (Fig. 1). Equilib-rium was reached after 30 min, and 25AM UDP-pyridoxalgaverapid and almost complete inhibition, which could not be over-come by high concentrations of UDP-glucose in the assay buffer.The overall equilibrium constant for reaction of UDP-pyridoxaland(1-.3)-ft-glucanIsynthase was measured as 0.84 ± 0.05 AM,this being the concentration of UDP-pyridoxal that gave 50%-inhibition of enzyme activity after incubation to equilibrium (see"Materials and Methods").The initial rate of decrease of enzyme activity during prein-

cubation was found to saturate with increasing concentrations ofUDP-pyridoxal, giving a maximal rate of 0.22 min-' at pH 7.0,with the half-maximal inactivation rate occurring at aKi of3.2 ± 0.5jsM (Fig. 2). It was necessary to determine these valuesat pH 7.0, when the rate was sufficiently low to measure accu-rately. The initial rate of reaction increased, although gradually,

c~ ~~~~ricbto ot °C(in

0

.580

a.2 60-0CP

40-

20

0 10 20 30 40 50 60

FIG. 1. Inhibition of(1-*3)-g1ucan synthase by reaction with UDP-pyridoxal. Membranes (56 ;&g) were incubated for the time shown in 801u 50 mm Hepes/KOH, 5mM MgCl2, 5 mm CaCI2, 10 mM cellobiose,0.1% digitonin (pH 7.3) containing zero (U), 1 uM (0), 5 $&M (0), or 25AM (A) UDP-pyridoxal. Triplicate 20,ul aliquots were then removed andassayed for 3 min in 1.0 mM UDP-[4JC]glucose as in "Materials andMethods." Control activities without preincubation were measured byassaying 14 jig membranes directly in zero, 0.1 Mm, 0.5 Mm, and 2.5;MmUDP-pyridoxal, these being the concentrations of inhibitor carried overinto the assay from the preincubated tubes.

25

20-

Initial rate / at min-'

of decrease . . 7

of enzyme 1 5 - )tactivity .7(%.min-')

Kinact= 3.2pM

4& ~~8 12 16

[UDP-pyridoxalJ IM in preincubationFIG. 2. Dependence of initial rate of reaction with UDP-pyridoxal on

concentration of UDP-pyridoxal. UDP-pyridoxal was added to the con-centrations shown to membranes (2404g) in 200 Md 50 mM Hepes/KOH,5mM MgC12, 5mM CaCl2,10 mM celiobiose, 0.16% digitonin, and at 20s intervals thereafter single 20 Ml aliquots were removed for assay for 3min in 1.0 mM UDP-['4CJglucose as in "Materials and Methods." Themeasured enzyme activities were plotted against time of preincubationfor each UDP-pyridoxal concentration, and the rates of decrease ofactivity measured and replotted against inhibitor concentration.

with increasing pH: for1 puM UDP-pyridoxal it was 2.7-fold fasterat pH 7.8 than at pH 7.0, and 5.8-fold faster at pH 8.6 than atpH 7.0. This variation was in the opposite manner to the pH-dependence of enzyme activity, which had an optimum at pH7.0, 64% of maximal activity at pH 7.8, and 15% at pH 8.6. Itwas therefore possible to study the reaction of UDP-pyridoxalwith(1l-.3)--glucan synthase by choosing conditions in which

1010 READ AND DELMER

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INHIBITION OF (1--3)-f,-GLUCAN SYNTHASE BY UDP-PYRIDOXAL

little inhibition occurred during the subsequent enzyme assay.Typically, membranes were incubated with UDP-pyridoxal atpH 7.8 and 0°C for various times before 10-fold dilution intoassay cocktail containing a high concentration of UDP-['4C]glucose, generally 1 mm, at pH 7.0 for a 3 min assay at 25°C.Under these conditions, less than 5% of the enzyme activity waslost during the assay.

Incubations of membranes with UDP-pyridoxal were per-formed in Hepes buffers, as it was found that Tris buffers had astrong protective effect on the enzyme. Similarly, polyamines,which activate (1--3)-fl-glucan synthase (13), could not be usedduring preincubation. These observations are consistent withUDP-pyridoxal acting as an inhibitor by reacting with aminogroups. Equally, UDP-pyridoxine, produced by treatment ofUDP-pyridoxal with NaBH4. was incapable of causing any time-dependent reduction in enzyme activity, indicating that thereaction required the aldehyde group. UDP-pyridoxine was, how-ever, a competitive inhibitor of enzyme activity, but with aconsiderably higher Ki (approximately 120 ,M) than that ofUDP-pyridoxal.

Inhibition by UDP-pyridoxal was reversed by dilution of themembranes, or by washing them free of inhibitor by centrifuga-tion twice for 30 min at 1 5,000g and resuspension in fresh buffer(not shown), but the easiest way to reactivate UDP-pyridoxylated(1--3)-fl-glucan synthase was to add cysteamine to 10 mm andincubate for 30 to 90 min at 0°C (Fig. 3). Subsequently assayedactivity returned in a time-dependent manner, with over 90%recovery after 90 min from enzyme treated with 5 uM UDP-pyridoxal. Cysteamine is an aminothiol and is capable ofreactingwith Schiff's bases to reform a free amino group and give a stablethiazolidine derivative of the aldehyde (3). Treatment of UDP-pyridoxylated membranes with NaBH4 completely preventedreactivation of the enzyme by centrifugation and resuspension,or by cysteamine (Fig. 3). NaBH4 at 50 Mm was sufficient to makeirreversible the inhibition caused by 5 Mm UDP-pyridoxal. Nei-ther NaBH4 nor cysteamine had any effect on enzyme activity

100

0

E

C1M

80

60

40

201

-o 5 10 15 ZO 25

[UDP-pyridoxol] UM

FIG. 3. Effect of cysteamine and borohydride on UDP-pyridoxalinhibition. Membranes (14.5 Mg) were incubated at 0°C for 30 min in150 50 mM Hepes/KOH, 5 mM MgCl2, 5 mM CaC12, 100 mm sucrose,0.01% digitonin (pH 7.8) containing UDP-pyridoxal as shown, followedin some cases by reduction (5 mM NaBH4 for 10 min) or reactivation(10 mm cysteamine for 90 min) treatments. UDP-['4C]glucose (20 M1 10mM) was then added to give a final volume of 200 Ml, and enzymeassayed for 3 min as in "Materials and Methods." Control tubes hadUDP-pyridoxal added simultaneously with UDP-['4C]glucose at the startof the assay. Treated with UDP-pyridoxal alone, (0); treated with UDP-pyridoxal then NaBH4, (0); treated with UDP-pyridoxal then cysteam-ine, (U); treated with UDP-pyridoxal then NaBH.. then cysteamine, (A).

in the absence of UDP-pyridoxal, but whenever NaBH4 reduc-tion was performed cellobiose was replaced by the nonreducingactivator sucrose.

This chemistry of inhibition is consistent with the reversibleformation ofa Schiffs base between the aldehyde group ofUDP-pyridoxal and an enzyme lysine residue(s), followed by reductionto e-N-UDP(pyridoxyl)-lysine.

Specificity of Reaction with UDP-Pyridoxal. The presence ofUDP-glucose during incubation of membranes with UDP-pyri-doxal gave substantial protection to subsequently assayed(1-*3)-,B-glucan synthase activity (Fig. 4), in a manner dependenton the concentration of UDP-glucose. This is consistent with thecompetitive kinetics ofinhibition that were observed when UDP-pyridoxal was added at the start of enzyme assays (see above).UDP and UDP-xylose are also competitive inhibitors of (1--3)-#-glucan synthase (not shown), but are incapable of forming anycovalent bond with the enzyme. When 1.5 mm UDP-xylose or1.0 mm UDP was included in preincubation of membranes withUDP-pyridoxal, little time-dependent modification of the en-zyme was observed (Fig. 4). These observations strongly suggestthat reaction of UDP-pyridoxal with (1--3)-fl-glucan synthaseoccurs at a UDP-sugar-binding site on the enzyme.

Reaction of membranes with UDP-pyridoxal was normallyperformed in the presence of the full set of activators of (1- 3)-f,-glucan synthase, namely 5 mM MgCl2, 5 mm CaCl2, 10 mMcellobiose or 100 mm sucrose, and digitonin (1.5 gg digitonin/,Mg protein). When the dependence of inhibition of enzymeactivity on the presence ofthese effectors was investigated, it wasfound that the reaction with UDP-pyridoxal had a strong require-ment for Ca2 . Ifthe modification were performed in the presenceof 1 mM EDTA or 1 mM EGTA, virtually no inhibition of theenzyme occurred, while a slow rate of inhibition was observed ifthe reaction was performed in 1 mM EGTA, 5 mm Mg2". Rapidinhibition by UDP-pyridoxal occurred in the presence of 5 mm

0 50C

06

0

CtCC

5 10 20 30

Preincubation ot OC in 5piN UDP-pyridoxal (min)FIG. 4. Protection against UDP-pyridoxal by substrate and competi-

tive inhibitors. Membranes (58 ug) were incubated at OC in 50 mmHepes/KOH, 5 mm CaC12, 5 mM MgC2, 10 mM cellobiose, 0.1%digitonin (pH 7.8) containing 5 ,uM UDP-pyridoxal and either no protec-tants (A), or 1.5 mm unlabeled UDP-glucose (0), or 3.0 mM unlabeledUDP-glucose (A), or 1.0 mM UDP (U), or 1.5 mM UDP-xylose (0). Afterthe times shown, triplicate volumes containing 14 Mg protein were

removed and diluted, usually 10-fold, into assay buffer containing a finalconcentration of 1.0 mM UDP-['4C]glucose, and assayed for 3 min as in"Materials and Methods." Control tubes contained enzyme assayed bysimultaneous addition of 1.0 mm UDP-['4C]glucose, 0.5 MM UDP-pyri-doxal, and where required appropriate concentrations of UDP or UDP-xylose, to allow for the inhibition by UDP-pyridoxal, UDP, and UDP-xylose carried over into the assay from the preincubated tubes.

a a a I I^I

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Plant Physiol. Vol. 85, 1987

Ca2", but addition of 5 mM Mg2" had no further effect on thisrate. This stimulation of the reaction by Ca2l, and by Mg2" onlyin the absence of Ca24, resembled the effects of these activatorson the affinity of (1-*3)-fl-glucan synthase for UDP-glucose (8),and suggested that the affinity of the enzyme for UDP-pyridoxalmight vary in a similar way.

Further evidence for this was provided by the interaction ofCa2+ and cellobiose in promoting the modification reaction.Figure 5 presents time-courses of inhibition of enzyme activityin the presence of 5 mM Mg2", and shows that Ca2" and cellobiosehave to be present together for maximal inhibition. When testedindividually, though, 5 mM Ca2" allows greater inhibition thandoes 10 mm cellobiose. This synergistic effect of Ca2" and cello-biose strongly resembles their effect on the affinity of (1- 3)-(#-glucan synthase for UDP-glucose (8). On the other hand, digi-tonin had no effect on the rate of inhibition by UDP-pyridoxalof ( 1--3)-fj-glucan synthase subsequently assayed in the absenceof digitonin (not shown), and this effector does not alter theaffinity of the enzyme for UDP-glucose (8). These data togethersuggest that the UDP-sugar-binding site that is modified by UDP-pyridoxal may well be the (l1- 3)-#-g1ucan synthase active site.

Incubation of membranes with pyridoxal-P in the presence ofCa2", Mg2+, and sucrose also resulted in a time-dependent lossof (1-.3)-fl-glucan synthase activity, but approximately 40-foldhigher concentrations of this inhibitor were needed compared toUDP-pyridoxal. (l1-3)-#-Glucan synthase was inhibited to19.8% residual activity by preincubation for 30 min in 250 AMpyridoxal-P at 0C, and this was readily reversed by treatmentwith 20mM cysteamine for 90 min (to 91.1% activity remaining);treatment of the pyridoxylated membranes with 5 mm NaBH4for 15 min before addition of cysteamine completely preventedthis reactivation (24.6% activity remained). Pyridoxine phos-phate gave no time-dependent inhibition. Thus, pyridoxal-P alsoinhibited (1--3)-f-glucan synthase by formation of a Schiff'sbase or bases.However, pyridoxal-P was not as specific in its modification

of(1l--3)-#3-g1ucan synthase as was UDP-pyridoxal. When addedsimultaneously with UDP-['4C]glucose in 3 min assays, pyri-doxal-P caused both an increase in Km and a decrease in Vm,

10

0

C

60

aE

05 20

&o Io1 20 iO 40 50 60

Preincubotion at OC in 5pM UDP-pyridoxol (min)FIG. 5. Requirement for Ca2" and cellobiose of reaction with UDP-

pyridoxal. Membranes (14.5 ,Ag) were incubated at 0'C in 168 Al Hepes/KOH, 5 mM MgC92, 0.1% digitonin (pH 7.8) containing 5 lM UDP-pyridoxal and either 1 mMEGTA (0), or 1 mmEGTA, 10 mmcellobiose(U), or 5 mMCaC12 (0), or 5 mmCaC12, 10mm cellobiose (A). After thetimes shown, CaCI2 and cellobiose were added as appropriate to 5 mmand 10mm final concentrations and UDP-['4C]glucose was added to 1.0mM, in a final volume of 200 Ml, and assays performed for 3 min as in"Materials and Methods."

indicating that inhibition was not simply competitive (notshown). Incubation of membranes for 20 min with 250 gMpyridoxal-P in the presence of 1 mm EDTA and no cellobiosestill caused 46% inhibition of (l-3)-#3-g1ucan synthase, whilethese conditions afforded almost complete protection against 5Mm UDP-pyridoxal. Similarly, 6 mM UDP-glucose, 3 mm UDP-xylose or 3 mm UDP were able to afford no more than 52%protection to enzyme activity on incubation with pyridoxalphosphate in the presence of Ca24 and cellobiose, while lowerconcentrations of these compounds conferred full protectionagainst inhibition by UDP-pyridoxal (Fig. 4). These data togetherimply that about half of the inhibition of (1--3)-f3-glucan syn-thase by pyridoxal phosphate is caused by formation of Schiffsbases with amino groups that are not at the active site of theenzyme. We conclude that the UMP moiety of UDP-pyridoxalis responsible for the high affilnity of this inhibitor for (1-43)-(3-glucan synthase, and that inhibition then occurs by reaction withan amino group at the enzyme active site.

Investigation of Membrane Sidedness using UDP-Pyridoxal.The above experiments were performed with membranes pre-pared in the absence ofosmoticum, stored at -80°C, and reactedwith UDP-pyridoxal and assayed in the presence of digitonin;the detergent stimulated their (1-+3)-fl-glucan synthase activity1.8- to 1.9-fold. Freshly prepared membranes, however, had thehighest degree of digitonin stimulation of synthase activity, gen-erally 4-fold to 5-fold with 1.5 ug digitonin/Mg protein. Centrif-ugation and resuspension of membranes, several sequentialfreeze-thawing cycles, or brief sonication had no effect on thetotal enzyme activity, but stimulated activity assayed withoutdigitonin and so progressively reduced the degree of digitoninstimulation observed (not shown). This reduction in the latencyof enzyme activity was considerably slower for membranes pre-pared and frozen-thawed in the presence of 250 mm sorbitol asosmoticum. (Latency is defined as that fraction of activity thatrequires digitonin for assay.) This similarity between the additionof digitonin and procedures that disrupt membrane integritysuggested that digitonin functioned as an enzyme activator bydisrupting permeability barriers in the membrane vesicles andcausing more enzyme active sites to be revealed to substrateUDP-glucose. We therefore tested the extent to which digitoninwas required for inhibition of latent enzyme activity by UDP-pyridoxal and NaBH4.Membranes were prepared from mung bean seedlings in the

presence of sorbitol, and were assayed for (1- 3)-#-glucan syn-thase activity with or without 0.03% digitonin; the enzyme wasstimulated 3.7-fold by the detergent. When samples were treatedin the absence of digitonin with sufficient UDP-pyridoxal andNaBH4 toinhibit almost completely the accessible enzyme, thenwashed free of inhibitors and assayed, enzyme assayed in theabsence of digitonin was found to have been inactivated by over90%, while latent activity had been inhibited by under 20%(Table I). This latent activity could be fully inhibited by UDP-pyridoxal in the presence of digitonin. These data are consistentwith the existence oftwo populations of(1l- 3)-#-glucan synthaseenzyme molecules, one set accessible to UDP-pyridoxal in theabsence of digitonin and another set that required detergent tobe modified.

Reaction of Polypeptides with UDP-[3HjPyridoxal. The reac-tion of mung bean membrane proteins with NaB3H4 was foundto give a background incorporation that was too high to permitidentification of polypeptides specifically labeled with UDP-pyridoxal and NaB3H4 (not shown), and so UDP-[3H]pyridoxalwas synthesized, purified, and its properties and reactivity veri-fied as in "Materials and Methods."When membrane proteins were solubilized in digitonin and

treated with 6 gM UDP-[3H]pyridoxal for 12 minin the presenceof various effectors, followed by reduction by 200 gM NaBH4,

1012 READ AND DELMER

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INHIBITION OF (1--3)-f-GLUCAN SYNTHASE BY UDP-PYRIDOXAL

Table I. Inhibition and Assay of(I - 3)-f3-Glucan Synthase in thePresence and Absence ofDigitonin

Freshly prepared membranes were incubated at 0.64 mg/ml at 0°C in400 1A 50 mm Hepes/KOH (pH 7.8), 5 mM MgCl2, 5 mm CaC12, 100mm sucrose, 100 mm sorbitol, with or without 0.1% digitonin, and eitherleft untreated or treated with 5 gM UDP-pyridoxal for 15 min, then 200lM NaBH4 for 15 min. Membranes were then centrifuged for 30 min at15,OOOg, washed in 1.0 ml 25 mm Hepes/KOH (pH 7.0), 1 mm EDTA,250 mm sorbitol with or without 0.03% digitonin as appropriate, recen-trifuged, resuspended in 400 ,ul of this buffer, and samples removed forassay. Assays contained 250 mm sorbitol and zero or 0.03% digitonin,but otherwise were as in "Materials and Methods."

Assay Specific LatentTreatment Conditions Activity Activity

nmol. min-' * mg-'protein

None No digitonin 19.0+ Digitonin 48.2 29.2

UDP-pyridoxal, No digitonin 1.8no digitonin + Digitonin 25.9 24.1

UDP-pyridoxal, + Digitonin 3.8digitonin

a

bc

l.O01. wt. 29

I t t t45 68 97 205

direction of electrophoresis

FIG. 6. Reaction of membrane polypeptides with UDP-[3H]pyri-doxal. Mung bean membrane proteins (72 Mg) solubilized in digitoninwere incubated at oeC in 240 Ml 50 mM Hepes/KOH, 5.6% glycerol,0.3% digitonin, containing 6 AM UDP-[3H]pyridoxal and various effec-tors, for 12 min at 0°C, then reduced with 200 Mm NaBH4 for 15 min at0°C. After triplicate 17 Ml aliquots were removed for assay, samples were

TCA-precipitated, and 5 gg of each was analyzed by SDS-PAGE andfluorography. The effectors were 5 mM CaC12, 100 mM sucrose (track a),or 5 mM MgC92, 1 mM EGTA (track b), or 2 mm EDTA (track c).(I-3)-f-Glucan synthase activities following modification and reductionwere, respectively, 21, 84, and 85% ofthat assayed with no preincubation.The vertical dotted line corresponds to a mol wt of 42,000.

the incorporation of radioactivity into 5 ,g of TCA-precipitableprotein ranged from 150 to 250 cpm, which corresponds to only60 to 100 nmol 3H/g protein. Substitution of UDP-[3H]pyridox-ine for UDP-[3H]pyridoxal, or 10 mm cysteamine for NaBH4,completely abolished this incorporation, implying that it was alldue to formation and reduction of Schiffs bases between UDP-[3H]pyridoxal and protein lysine residues. Analysis of labeledpolypeptides by SDS-PAGE, fluorography and densitometricscanning showed that similar sets of polypeptides were labeledwith UDP-[3H]pyridoxal under a variety of conditions (Fig. 6).Track a shows the polypeptides labeled under conditions wherethe enzyme activity was irreversibly inhibited (5 mM CaCl2, 100mM sucrose), while tracks b and c resulted from modification

under conditions where the enzyme activity was protected (5 mMMgCl2, 1 mm EGTA, track b, or 2 mM EDTA, track c). Theabsence ofany unique polypeptide labeled only under conditionsthat irreversibly inhibit (1-+3)-fl-glucan synthesis (i.e. conditionsused for track a) in this and many other similar experiments,means that the enzyme catalytic polypeptide was not identified.However, a polypeptide of mol wt 42,000, marked in Figure 6,was found to incorporate radioactivity from UDP-[3H]pyridoxalspecifically in the presence of divalent cations (tracks a and b);the labeling of this polypeptide was found to be greater inreactions containing 5 mM CaC12 plus 5 mM MgCl2 than inreactions containing 5 mM CaCl2 alone (not shown).

Digitonin-solubilized proteins were enriched for (1--3)-#3-glu-can synthase by glycerol gradient centrifugation followed byanion-exchange FPLC. The degree of purification was at least40-fold but could not be reliably quantitated because the enzymeactivity was unstable. SDS-PAGE analysis of the polypeptideseluting in fractions across the peak of (1--3)-ft-glucan synthaseactivity from the FPLC column is shown in Figure 7. Fractions18 to 21, eluting at 0.12 to 0.15 M NaCl, contained the peakactivity, and these were pooled, treated with UDP-[3H]pyridoxal,then reduced with NaBH4, precipitated with TCA, and analyzedby SDS-PAGE and fluorography. The major polypeptide labeledwas at mol wt 42,000 (Fig. 7) and, as in unfractionated prepara-tions, reaction of this polypeptide with UDP-[3H]pyridoxal re-quired only an excess of divalent cation (either Mg2" alone orwith Ca2"); once again, no polypeptide was labeled only underconditions (presence of CaC12 plus sucrose) that inhibited(1-.3)-f3-glucan synthase activity. Digitonin-solubilized mungbean membranes contain at least 20 polypeptides that react withUDP-[3H]pyridoxal (Fig. 6). Partial purification of (1- 3)-#3-glucan synthase strongly enriched for one of these reactive poly-peptides, at a mol wt of42,000, which suggests that this polypep-tide may play some role in glucan synthesis.

DISCUSSIONIn the absence of purified preparations of (1--3)-f,-glucan

synthase, it is necessary to seek information about the proteincomponents of this enzyme from studies on unfractionatedmembranes. The simple kinetics and chemistry of inhibition of(l1--3)-f-glucan synthase by UDP-pyridoxal justified its use un-der these conditions. Reaction of the enzyme with UDP-pyri-doxal occurred rapidly even at 0WC, and from the equilibriumposition reached a Ko of0.84 ± 0.05 AM was calculated, implyingthat the enzyme has a high affinity for this inhibitor. Inhibitedenzyme could be reactivated by washing the membranes free ofinhibitor or by addition of the aminothiol cysteamine, whiletreatment with borohydride made the inhibition irreversible.These observations, and the lack of reactivity of ( 1- 3)-fl-glucansynthase with the alcohol UDP-pyridoxine, strongly suggest thatinhibition occurs by formation of a Schifis base between thealdehyde group of UDP-pyridoxal and an amino-group on theenzyme, as was found for reaction of UDP-pyridoxal with gly-cogen synthase (27) and as is generally the case for modificationof enzymes with pyridoxal-P (1 1). It is therefore probable that(1--3)-f3-glucan synthase contains an essential lysine residue orresidues.The affinity of (1--3)-fl-glucan synthase for UDP-pyridoxal

was high even under conditions where this compound was actingonly as a competitive inhibitor: a Ki of 3.8 ± 0.7 AM was foundin brief (3 min) assays at pH 7.8 in the presence of varyingconcentrations of UDP-glucose. This value was similar to theKi. found for reaction of UDP-pyridoxal with (1-+3)-,3-glucansynthase during preincubations at pH 7.0 (3.2 ± 0.5 AM), whichsuggests that the same step could be rate-limiting in both cases;this step may, however, not represent the formation of a non-covalent complex ofenzyme and inhibitor before reaction, since

1013

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Plant Physiol. Vol. 85, 1987

dbelIed X resenc eXo.

M g m1gy <'>2 v, S,*\. i . V 1

traction no.

: .". ;" ~ , 24 ..& r t is

1.

9' jI-S

FIG. 7. Co-purification of the m.w. 42,000UDP-pyridoxal-reactive polypeptide with (1--3)-,B-glucan synthase activity. FPLC column frac-tions eluted with a gradient of increasing NaClconcentration were pooled as shown, and portionswere TCA-precipitated and analyzed by SDS-PAGE, staining with Coomassie blue R. Protein(10 Mg) from fractions 18 to 21 was incubated to280 Ml 50 mm Hepes/KOH, 3.6% glycerol, 0. 18%digitonin, plus effectors as shown, containing 7AM UDP-[3H]pyridoxal, for 20 min at OC, thenreduced with 333 AM NaBH4 for 20 min, TCA-precipitated, and analyzed by SDS-PAGE andfluorography. Effector concentrations in the mod-ification reaction were 5 mM MgCl2, 1 mm EGTA,or 5 mM MgCl2, 5 mm CaCl2, 100 mm sucrose.

ooumassie staininng

r;1tiortS troirn F

A rOri} f.-V han ge (loiiu rrh?

it would be expected that this noncovalent binding constant forUDP-pyridoxal would not be much different from that for UDP-pyridoxine (which cannot participate in a chemical reaction withan amino group), measured at 120 ,M. It is possible thereforethat the low Ki for UDP-pyridoxal is due to rapid and reversibleaddition onto an amino group to form a carbinolamine, with aformation constant of 3 to 4 ,M, and that this then undergoesslow dehydration to the Schiff's base. Since the reaction was

performed with total membrane preparations, rather than a

purified protein, it was not possible to test this hypothesis byfollowing the course ofthe reaction with (I-3)-(3-glucan synthasespectrophotometrically. In any case, the high affinity of UDP-pyridoxal for (1--3)-o-glucan synthase meant that it was verysuitable as a labeling reagent for this enzyme. Tagaya et al. (27)also found that the affinity of glycogen synthase for UDP-pyridoxal (Kin,,, of 25 uM) was apparently higher than that forsubstrate UDP-glucose (Km of 120 Mm).The chemistry of inhibition of (1- 3)-f-glucan synthase by

pyridoxal-P indicated that this too occurred by formation ofSchiff's bases with protein amino groups, but UDP-pyridoxalwas a considerably more useful inhibitor not just by virtue of its40-fold higher affinity but also because of its greater specificityof reaction. Even high levels of UDP-glucose were incapable ofprotecting more than halfofthe ( 1--3)-j3-glucan synthase activityagainst inhibition during reaction with pyridoxal-P, but UDP-glucose, UDP-xylose and UDP completely protected the enzymeagainst modification with UDP-pyridoxal. Similarly, the reactionwith UDP-pyridoxal was inhibited over 90% by 1 mM EDTA or1 mm EGTA, while up to 50% of the inhibition caused byreaction with pyridoxal-P occurred at lysine residues not requir-ing Ca2l for modification and therefore presumed not to belocated at the active site. This higher specificity of reaction withUDP-pyridoxal concurs with findings on modification by nucle-oside-substituted pyridoxal phosphates of some nucleotide-bind-ing enzymes (28) and the nucleotide-sugar-binding enzyme gly-cogen synthase (27): addition of the nucleoside moiety gives theinhibitor a higher affinity for the enzyme active site, allowinglower concentrations to be used, which prevents reaction withamino groups elsewhere in the protein. This also agrees well withthe direct evidence for modification of ( 1--3)-o-glucan synthase

at its active site, the observation that the enzyme activatorsrequired for reaction with UDP-pyridoxal (Ca2" and, less effec-tively, cellobiose or sucrose) were those activators that stimulate(I-3)-#-glucan synthesis by increasing the affinity ofthe enzymefor UDP-glucose (8).The mechanism of stimulation by digitonin could possibly

include inhibition ofthe competing synthesis ofsteroyl glucosidesfrom UDP-glucose, direct activation of the enzyme, or permea-

bilization of membrane vesicles to UDP-glucose (9, 14, 30).Inhibition ofthe synthesis of steroyl glucosides would not explainthe observation that the degree of digitonin stimulation is inde-pendent of UDP-glucose concentration from 0.1 to 1.0 mm, andthe experiments described here were designed to distinguish theother alternatives. UDP-pyridoxal, like UDP-glucose, would notbe expected to cross intact membranes, and can be used to reactwith amino groups revealed only at one side of a membrane;pyridoxal phosphate has been used similarly (20). Table I showsclearly that a certain amount of enzyme activity is protectedfrom UDP-pyridoxylation by the absence of digitonin, and thatthis is close to the amount by which enzyme activity is increasedby the addition of digitonin. The ability of digitonin to reveal apopulation of enzyme molecules to UDP-pyridoxal is consistentwith the detergent stimulating total (1- 3)-fl-glucan synthaseactivity by permeabilizing vesicles, and allowing UDP-glucose toenter those in which the enzyme active site is on the inside.The high affinity, specificity, and irreversible nature of the

modification of (1--3)-fl-glucan synthase with UDP-pyridoxaland borohydride suggested the possibility of identifying the cat-alytic polypeptide of this enzyme in unfractionated membranesby reaction with UDP-[3H]pyridoxal. Candidate polypeptideswould incorporate radioactivity under conditions that wouldspecifically allow inhibition of the enzyme (5 mm CaCl2, 100mm sucrose), but not when the enzyme was protected frommodification (1 mM EGTA). No such polypeptide was found bySDS-PAGE, even after the reaction was performed under a widevariety of conditions, using intact membranes or digitonin-solu-bilized material. All the incorporated radioactivity was howevershown to result from reduction of Schifis bases formed fromUDP-[3H]pyridoxal and protein amino groups, and Tamura etal. (28) have shown that a similar labeling procedure using ADP-

Huorogyami-i;, ) .p-' H vatef

..~f' t r t'" fir ctxF on s s S

1014 READ AND DELMER

1-

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INHIBITION OF (1--3)-fl-GLUCAN SYNTHASE BY UDP-PYRIDOXAL

[3H]pyridoxal works well with some purified proteins. In caseinsufficient enzyme protein were present in these total membranepreparations, membrane proteins were dissolved in digitonin andenriched for (l1- 3)-fl-glucan synthase by glycerol gradient cen-trifugation and anion-exchange FPLC, but even this partiallypurified preparation incorporated no radioactivity from UDP-[3H]pyridoxal specifically in the presence of Ca2l and sucrose.Wasserman and McCarthy (30) have shown that depletion ofboundary-layer lipids can cause loss of (l1--3)-#-glucan synthaseactivity, and it may be that the ability to bind UDP-pyridoxal isalso progressively lost during purification in a similar way. Twoother possible nontrivial explanations for lack of specific reactionwith UDP-[3H]pyridoxal are that the reactive enzyme aminogroup may not be a lysine residue but may be on a dissociableco-factor or lipid and, second, that the enzyme may not beprecipitated by 12.5% TCA or enter or focus in a SDS-PAGEgel by virtue of its properties being altered by covalently attachedglucan molecules.

However, one polypeptide was detected that was specificallylabeled by UDP-[3H]pyridoxal. The reaction of this polypeptide,of mol wt 42,000, with UDP-[3H]pyridoxal was stimulated byMg2" or Ca2", and strikingly it was the major polypeptide ofthose capable of being labeled that co-purified with (1- 3)-(3-glucan synthase activity. We have previously identified a mungbean membrane polypeptide of very similar mol wt, measuredas 44,000, as an acceptor for transfer of radioactivity from UDP-[14C]glucose in the presence of Mg2" or Mn2" (25), this labelingbeing presumed to represent glycosylation. Even more interest-ingly, glycosylated polypeptides of very similar sizes (mol wt

38,000-42,500) have also been identified in several other labo-

ratories, and have been postulated to be involved in the synthesisof glucan (22), arabinan (7), or arabinoprotein (2) in membranesfrom leguminous plants, and starch in potato tubers (18). It is

possible that one or more of these glycosylated polypeptides isthe same species as reacts here with UDP-[3H]pyridoxal, that itis involved in some way, as a primer, carrier or intermediate, inthe synthesis of (1-63)-j-glucan, and that this polypeptide maybe associated with the glucan synthase complex. Whether or notthis is the case, these studies show that nucleoside-diphospho-pyridoxal derivatives will be useful tools in investigating theproperties of polysaccharide synthases.

Acknowledgments-We are very grateful to Dr. Toshio Fukui of Osaka Univer-

sity, Japan, for his kind gift of UDP-pyridoxal, to John Bussell for assistance in

purification of (1-.3)-fl-glucan synthase, and to all the members of the ARCO

Plant Cell Research Institute (now PCRI Inc.) for stimulating discussion.

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6. EIBERGER LL, K MCCARTHY, BP WASSERMAN 1987 Partial purification of

digitonin-solubilized ,B-glucan synthase from red beet root. Plant Physiol 83:

982-9877. HAYASHI T, G MACLACHLAN 1984 Biosynthesis of pentosyl lipids by pea

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DELMER 1987 UDP-Glucose: (1-63)-glucan synthases from mung beanand cotton: differential effects of Ca2l and Mg2" on enzyme properties, andon macromolecular structure of the glucan product. Plant Physiol 83: 1054-1062

9. HENRY RJ, BA STONE 1982 Solubilization of ,-glucan synthases from themembranes of cultured ryegrass endosperm cells. Biochem J 203: 629-636

10. JAFFE MJ, AC LEOPOLD 1984 Callose deposition during gravitropism of Zeamays and Pisum sativum and its inhibition by 2-deoxy-D-glucose. Planta161: 20-26

11. KARPEISKY MY, HBF DIXON 1986 The use of pyridoxal phosphate for modi-fication of proteins. In D Dolphin, R Poulson, 0 Avramovic, eds, VitaminB6, Pyridoxal Phosphate: Chemical, Biochemical and Medical Aspects.Wiley, New York, pp 71-116

12. KAuss H 1987 Some aspects of calcium-dependent regulation in plant metab-olism. Annu Rev Plant Physiol 38: 47-72

13. KAuss H, WJEBLICK 1986 Synergistic activation of(I--3)-I#-D-glucan synthaseby Ca2 and polyamines. Plant Sci 43: 103-107

14. KAuss H, W JEBLICK 1986 Influence of free fatty acids, lysophosphatidylcho-line, platelet-activating factor, acylcarnitine, and Echinocandin B on 1,3-,B-D-glucan synthase and callose synthesis. Plant Physiol 80: 7-13

15. KOGA PG, RL CRoss 1982 Pyridoxylation of essential lysine residues ofmitochondrial adenosine triphosphatase. Biochim Biophys Acta 679: 269-278

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17. LASKEY RA, AD MILLS 1975 Quantitative film detection of 3H and 14C inpolyacrylamide gels by fluorography. Eur J Biochem 56: 335-341

18. MORENO S, CE CARDINI, JS TANDECARZ 1986 a-Glucan synthesis on a proteinprimer, uridine diphosphoglucose: protein transglucosylase, 1. Separationfrom starch synthase and phosphorylase and a study of its properties. Eur JBiochem 157: 539-545

19. MORROW DL, WJ LUCAS 1986 (I-.3)-(#-D-Glucan synthase from sugar beet I.Isolation and solubilization. Plant Physiol 81: 171-176

20. OHKAWA I, RE WEBSTER 1981 The orientation of the major coat protein ofbacteriophage fl in the cytoplasmic membrane of Escherichia coli. J BiolChem 256: 9951-9958

21. PARSONS TF, J PREISS 1978 Biosynthesis of bacterial glycogen. Isolation andcharacterization of the pyridoxal-P allosteric activator site and the ADP-glucose-protected pyridoxal-P binding site ofEscherichia coli B ADP-glucosesynthase. J Biol Chem 253: 7638-7645

22. QUENTMEIER H, E INGOLD, HU SEITz 1986 Purification and characterizationof an autocatalytic protein-glycosylating enzyme. In B Vian, D Reis, RGoldberg, eds, Proceedings of the Fourth Cell Wall Meeting, Paris, 1986.Groupe Parois, France, pp 372-373

23. RAY PM 1979 Maize coleoptile cellular membranes bearing different types ofglucan synthetase activity. In E Reid, ed, Methodological Surveys in Bio-chemistry, Vol 9, Plant Organelles. Ellis Horwood Ltd, Chichester, England,pp 135-146

24. READ SM. DH NORTHCOTE 1981 Minimization of variation in the response todifferent proteins of the Coomassie blue G dye-binding assay for protein.Anal Biochem 116: 53-64

25. READ SM, M THELEN, DP DELMER 1986 Identification of UDP-glucose-binding proteins in mung bean membranes. In B Vian, D Reis, R Goldberg,eds, Proceedings of the Fourth Cell Wall Meeting, Paris, 1986. GroupeParois, France, pp 308-311

26. STOCK A, F ORTANDERL, G PFLEIDERER 1966 Darstellung von radioaktivmarkiertem Pyridoxal-5'-phosphat. Biochem Z 344: 353-360

27. TAGAYA M, K NAKANO, T FUKUI 1985 A new affinity labeling reagent for theactive site of glycogen synthase. Uridine diphosphopyridoxal. J Biol Chem260: 66706676

28. TAMURA JK, RD RAKOV, RL CRoss 1986 Affinity labeling of nucleotide-binding sites on kinases and dehydrogenases by pyridoxal 5'-diphospho-5'-adenosine. J Biol Chem 261: 4126-4133

29. THELEN MP, DP DELMER 1986 Gel-electrophoretic separation, detection andcharacterization of plant and bacterial UDP-glucose glucosyltransferases.Plant Physiol 81: 913-918

30. WASSERMAN BP, KJ MCCARTHY 1986 Regulation of plasma membrane jB-glucan synthase from red beet root by phospholipids. Reactivation of TritonX-100 extracted glucan synthase by phospholipids. Plant Physiol 82: 396-400

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