udp-glucose: (1,3)-,@-glucan (callose) synthase l. entrapment' · andinsufficient detergentin...

Plant Physiol. (1991) 97, 684-6920032-0889/91 /97/0684/09/$01 .00/0

Received for publication December 7, 1990Accepted June 7, 1991

Rapid Enrichment of CHAPS-Solubilized UDP-Glucose:(1,3)-,@-Glucan (Callose) Synthase from Beta vulgaris L.

by Product Entrapment'

Entrapment Mechanisms and Polypeptide Characterization

Ayong Wu, Robert W. Harriman, David J. Frost, Stephen M. Read2, and Bruce P. Wasserman*

Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University,New Brunswick, New Jersey 08903-0231

ABSTRACT

Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-#-glucan (callose) synthase from storage tissue of red beet (Betavulgaris L.) is obtained when the preparation is incubated with anenzyme assay mixture, then centrifuged and the enzyme releasedfrom the callose pellet with a buffer containing EDTA and CHAPS(20-fold purification relative to microsomes). When centrifuged athigh speed (80,000g), the enzyme can also be pelleted in theabsence of substrate (UDP-Glc) or synthesis of callose, due tononspecific aggregation of proteins caused by excess cationsand insufficient detergent in the assay buffer. True time-depend-ent and substrate-dependent product-entrapment of callose syn-thase is obtained by low-speed centrifugation (7,000-11,000g) ofenzyme incubated in reaction mixtures containing low levels ofcations (0.5 millimolar Mg2+, 1 millimolar Ca2 ) and sufficientdetergent (0.02% digitonin, 0.12% CHAPS), together with cello-biose, buffer, and UDP-Glc. Entrapment conditions, therefore, area compromise between preventing nonspecific precipitation ofproteins and permitting sufficient enzyme activity for callosesynthesis. Further enrichment of the enzyme released from thecallose pellet was not obtained by rate-zonal glycerol gradientcentrifugation, although its sedimentation rate was greatly en-hanced by inclusion of divalent cations in the gradient. Prepara-tions were markedly cleaner when product-entrapment was con-ducted on enzyme solubilized from plasma membranes isolatedby aqueous two-phase partitioning rather than by gradient cen-trifugation. Product-entrapped preparations consistently con-tained polypeptides or groups of closely-migrating polypeptidesat molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27kilodaltons. This polypeptide profile is in accordance with thefindings of other callose synthase enrichment studies using avariety of tissue sources, and is consistent with the existence ofa multi-subunit enzyme complex.

This research was supported in part by grants from the U.S.Department of Agriculture (87-CRCR-1-2414), National ScienceFoundation (DCB-8907202), the Charles and Johanna Busch Foun-dation, and the New Jersey Agricultural Experiment Station withState and Hatch Act Funds. New Jersey Agricultural ExperimentStation, Publication No. D- 10546-1-90.

2 Present address: Plant Cell Biology Research Centre, School ofBotany, The University of Melbourne, Parkville 3052, Australia.

684

The procedure known as product entrapment has recentlygained widespread application for the rapid and straightfor-ward enrichment of solubilized polysaccharide synthases. Itsuse has been reported for purification of chitin (13) andcellulose synthase (15), and for partial purification of red beet(11) and mung bean (12) CSs.3 The procedure consists ofincubating solubilized enzyme with substrate (UDP-GlcNAcfor chitin synthase or UDP-Glc for cellulose and callosesynthase) under conditions that allow synthesis of insolublepolymeric product. Enzyme is thought to become trappedwithin or bound to the resulting meshwork of microfibrils,and is recovered in concentrated form by centrifugation. Thepolysaccharide synthase can then be released and the proce-dure repeated.

In the course of our work on CS (1 1), however, it becameclear that sedimentation of activity and concomitant purifi-cation need not actually require formation of any glucanproduct, but could also occur simply from precipitation ofthe enzyme in the assay mixture (which differs in compositionfrom the medium used to solubilize the enzyme). This paperdescribes the contribution of individual parameters (concen-trations of detergent, divalent cations and substrate, andcentrifugation force) to the insolubilization of CS, and dem-onstrates that true product-dependent sedimentation of CS isobtained only when modified reaction mixtures are centri-fuged at low speed. Furthermore, polypeptide profiles ofenriched CS fractions prepared by entrapment and otherpurification protocols are compared, and similarities are high-lighted.

MATERIALS AND METHODSMaterialsRed beets (Beta vulgaris L.) were obtained from local

markets. UDP-['4C]Glc (specific activity 220 mCi mmol-')

3 Abbreviations: CS, callose synthase; CHAPS, 3-[(3-cholamido-propyl)dimethylammonio]-1-propane sulfonate; P1, S1, first pellet,first supernatant from first sedimentation step; P2, S2, second pellet,second supernatant, from resuspension and centrifugation of firstpellet P1; PM, plasma membrane; Tris/HCl, tris[hydroxymethyl]aminomethane hydrochloride; UDP-Glc, uridine diphosphate glu-cose.

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CALLOSE SYNTHASE ENTRAPMENT AND POLYPEPTIDE CHARACTERIZATION

was a product ofICN Radiochemicals (Irvine, CA). Digitoninwas purchased from ICN Biochemicals (Cleveland, OH). Allother chemicals were purchased from Sigma Chemical Co.

Preparation of Membranes and Solubilized CS

Microsomal membranes were isolated from red beet storagetissue by differential centrifugation and PMs by centrifugationon discontinuous sucrose gradients as before (25). CHAPS-solubilized CS was prepared in 50 mm Tris/HCl, pH 7.5, 1mM EDTA, 1 mM EGTA, 0.6% CHAPS, and 7.5% glycerolby the two-step solubilization procedure as described (23, 25).Where indicated, PMs were also prepared by aqueous two-phase partitioning ( 18).

CS Assay

Assays were performed in a final volume of 100 ,uL con-taining enzyme and 5 mM MgCl2, 2 mm CaCl2, 5 mm cello-biose, 0.01% digitonin, 1 mm UDP-['4C]Glc (0.12 mCi/mmol), and 50 mm Tris/HCl, pH 7.5. Assays were conductedfor 15 min at 30°C and ethanol-insoluble glucan was measured(25). One unit of activity is defined as that which catalyzesthe incorporation of 1 nmol glucose min-' into ethanol-insoluble glucan.

Protein Determination

Protein was determined by Coomassie brilliant blue dye-binding with BSA as standard (2).

Product Entrapment Procedure

In the standard procedure adopted for obtaining time- andsubstrate-dependent product entrapment, reaction mixturesranging from 1.0 to 30 mL were prepared by combining 1volume of solubilized enzyme (0.2-0.4 mg protein mL-') with4 volumes of an effector mix to give final concentrations of:1 mM CaCl2, 0.5 mM MgCI2, 0.2 mm EDTA, 0.2 mm EGTA,0.02% digitonin, 5 mM cellobiose, 1.5% glycerol, and 0.12%CHAPS in 50 mM Tris/HCl, pH 7.5 (EDTA, EGTA, glycerol,and CHAPS levels reflect carryover from the solubilizationbuffer). Synthesis of glucan was initiated by addition of 1 mMUDP-Glc. After 40 min at 30°C, the reaction was stopped bycooling to 4°C for 30 min and the mixture was then centri-fuged at low speed (7,000g for 40 min at 4°C in a Sorvall SA-600 rotor or 11,000g for 40 min at 8°C in a Fisher microcen-trifuge). The supernatant (SI) was carefully removed, and thepellet (P1) resuspended in 0.5 to 15 mL of 0.1% CHAPS, 3mM EDTA, 10% glycerol, and 50 mM Tris/HCl, pH 7.5, andrecentrifuged at 7,000 or 11 ,000g for 40 min. The supernatant(S2) was carefully withdrawn and the pellet (P2) was resus-pended as above but with 0.05% CHAPS. Each fraction wasassayed for enzyme activity and protein. Pellets were mostefficiently resuspended by sonicating for several seconds in 40to 50 gtL of resuspension buffer before bringing to the finalvolume.

Glycerol Gradient Centrifugation

Linear gradients were made by combining 1.75 mL each of28% (w/w) and 80% (w/w) glycerol containing 0.1% CHAPS,

50 mM Tris/HCl, pH 7.5, on a gradient maker. Either divalentcations (1 mM CaC12, 5 mM MgCl2) or chelators (3 mM EDTA,3 mm EGTA) were also present. Samples of 1.2 mL ofreleasedenzyme (supernatant S2 in 10% glycerol) were layered on thegradients, which were then centrifuged at 160,000g for 1 hr.After centrifugation, a micropipet was carefully lowered tothe bottom of the centrifuge tube and the gradients werefractionated into eight fractions of 0.6 mL each. The pellets(which were not visible) were resuspended in 0.6 mL of0.05%CHAPS, 3 mM EDTA, 10% glycerol, and 50 mm Tris/HCl,pH 7.5. Fractions were assayed for enzyme activity, protein,and refractive index.

SDS-PAGE

Electrophoresis was performed on 9 to 18% SDS-PAGEgradients (14), with modifications (20). Polypeptides werevisualized by staining with Coomassie brilliant blue and silver(Daiichi Protocol, Integrated Separation Systems, Enprotech,Hyde Park, MA).

Excision of Polypeptides and Antibody Production

Preparative electrophoresis under denaturing conditionswas performed on 3 mm polyacrylamide gradient gels (9-18%) as described (11). Polypeptides were excised and elec-troeluted in an Elutrap (Schleicher and Schuell, Keene, NH).The protein was concentrated by precipitation with 25% (v/v) TCA. The pellet was washed three times with acetone toremove residual TCA and resuspended in 10 mm sodiumphosphate, pH 7.4, 140 mM NaCl (saline) containing 0.1%SDS. The eluted protein was incubated at 80°C for 10 min insaline with 0.1% SDS and 350 qL (100 ,ug), combined withan equal volume of Freund's complete adjuvant (Sigma), andinjected intradermally/subcutaneously into a New ZealandWhite rabbit. Antiserum was collected at 4 and 7 weeks afterthe first injection and partially purified according to Biggs etal. (1).

Western Blotting

PM proteins were electrophoresed on 1.5 mm gels andtransferred to nitrocellulose filters (Schleicher and Schuell)with a Transphor Electrophoresis Unit (Hoefer) for 1.5 h. Theblot was washed for 15 min in 150 mm NaCl, 0.5% Tween20, and 10 mm Tris-HCl, pH 8.0, and remaining sites wereblocked with the same buffer containing 0.2% casein. Theblot was incubated with antibodies in blocking buffer for 1 hat room temperature, washed three times for 10 min each,and was then incubated with alkaline phosphatase-conju-gated, anti-immunoglobulin G antibodies (Sigma) for 30 min.After washing three times in blocking buffer, the blot wasdeveloped by an alkaline phosphatase-mediated reaction (Pro-mega, Madison, WI).

Immunoinhibition

Antibodies directed against the 54- and 52-kD proteins atvarious dilutions were incubated with PMs for 30 min atroom temperature in 90 ,uL of 2.2 mM CaCI2, 5.5 mM MgCl2,5.5 mM cellobiose, 0.11% digitonin, and 54 mM Tris/HC1,

685



Plant Physiol. Vol. 97, 1991

pH 7.5. CS reactions were initiated by addition of 10 ,uL ofUDP-['4C]Glc (0.12 mCi mmol-'), and assayed as above.

RESULTS

Characterization of the Product EntrapmentProcedure. Precipitation of CS by High-SpeedCentrifugation in the Absence of UDP-Glc

Product entrapment was initially attempted ( 11) by prepar-ing and incubating standard CS assay mixtures followed bycentrifugation at high speed (80,000g, 40 min), similar to theapproaches used previously (12, 15). Extraction of the pellet(P1) with a buffer containing 3 mM EDTA, 0.1% CHAPS,and 10% glycerol, followed by low speed centrifugation(11 ,000g), resulted in release of enzyme into the supernatantS2 (Table I). Control mixtures, lacking UDP-Glc, were alsoprepared.The results show that, under these conditions, CS activity

is found first in the 80,000g pellet (P1), and is then releasedinto the subsequent 1 ,OOOg supernatant (S2). Typically, spe-cific activities are increased by three- to fourfold with goodyields, 51% in the experiment shown here (Table I). Thisrepresents a 20-fold enrichment relative to microsomal mem-branes (average specific activity 50 units/mg). Significantly,this purification procedure worked as well or better withcontrol samples that did not contain UDP-Glc, in whichformation of an insoluble product was not possible. Thepresence ofglucan plus divalent cations caused all the enzymeactivity to sediment into P1 (102% compared with 83%), butimpeded its resolubilization into S2 (50% versus 84%).CS and other proteins, therefore, can be pelleted from assay

Table I. Enrichment of CS by High-Speed Sedimentation in thePresence and Absence of SubstrateTwo incubation mixtures of 4.0 mL were prepared and 1 mm UDP-

Glc was added to one to initiate product formation. Each mixturecontained solubilized enzyme (0.8 mL, 0.336 mg), 1 mm CaCI2, 5 mmMgCI2, 0.2 mm EDTA, 0.2 mm EGTA, 1.5% glycerol, 5 mm cellobiose,0.01% digitonin, and 0.12% CHAPS in 50 mm Tris/HCI, pH 7.5, withaddition of UDP-Glc only where shown below. After 40 min at 300C,the reaction was stopped by cooling to 40C for 30 min, and thencentrifuged at 80,000g for 40 min. CS was released from the firstpellet (P1) as described in Materials and Methods. Numbers in paren-theses indicate enzyme yield after the second centrifugation relativeto the activity of the first pellet (P1).

Fraction Total Total Specific Purification Yield ofActivity Protein Activity Activityunits mg units mg-' -fold %

Solubilized enzyme 119.1 0.336 354 1 100No UDP-Glc

S1 24.6 0.137 178 0.5 21P1 99.3 0.145 68 1.9 83S2 83.1 0.067 1243 3.5 70 (84%)P2 27.0 0.090 300 0.85 23 (27%)

1 mM UDP-GlcSi 12.8 0.141 90 0.25 1 1P1 121.6 0.146 833 2.4 102S2 60.3 0.054 1117 3.1 51 (50%)P2 44.0 0.090 489 1.4 37 (36%)

mixtures at high centrifugal force independent of the forma-tion of callose. To distinguish between this precipitation andproduct entrapment (which must show a dependence on bothconcentration of substrate and time), factors influencing en-zyme solubility were varied, including centrifugation speed,cation concentration, and detergent levels.

Effect of Centrifugation Speeds

Lowering of centrifugation speeds was the most effectiveway to reduce glucan-independent precipitation from reactionmixtures. Sedimentation of newly synthesized glucan, pro-duced by incubating solubilized enzyme with UDP-['4C]Glc,was complete at 7000g, whereas almost no precipitation ofactivity (5% or less) occurred if solubilized enzyme was in-cubated in assay mixture lacking UDP-Glc, followed by cen-trifugation at 7000g for 60 min (not shown). At 11 ,OOOg for40 min, the precipitation of activity was higher and morevariable (10-20%).

Centrifugation of reaction mixtures over a glycerol cushiondid not reduce the extent of nonspecific precipitation.

Effect of Reaction Mixture Composition

CS is one of a group of PM proteins solubilized by 0.6%CHAPS in the presence of 1 mm EDTA and 1 mm EGTA;CHAPS does not remove the enzyme from the membranesin the presence of divalent cations (23). The assay mixture asoriginally formulated contains 0.12% CHAPS, 5 mM MgC12,and 2 mm CaCl2, and this lower detergent content and thereaddition of divalent cations could readily promote enzymeaggregation and insolubilization. Therefore, we attempted toreduce the nonspecific precipitation of CS in product-entrap-ment reactions by increasing CHAPS levels and reducing thedivalent cation concentration.

Increasing the CHAPS concentrations of entrapment mix-tures progressively inhibited callose formation, with completeenzyme inactivation occurring at 0.4% CHAPS (Fig. 1). Asimilar effect was shown with CS solubilized from celerypetioles (not shown). Protection against this inactivationcould not be obtained by maintaining a constant ratio ofCHAPS to digitonin (Fig. 1). The CHAPS concentration inproduct-entrapment reactions, therefore, was not increasedabove 0. 12%.Two assay mixture components, other than UDP-Glc, that

were found to promote precipitation ofthe solubilized enzymewere digitonin and the divalent cations Mg2' and Ca2+. Fig-ure 2 shows the effect of digitonin on the amount of enzymefound in the pellet (P1) following centrifugation at 1 1,000g.In the absence of UDP-Glc, a small amount of enzymeprecipitation occurred at low concentrations of digitonin(0.0025-0.005%), but this was prevented by 0.02% digitonin.In the presence of UDP-Glc, callose synthesis is stimulatedseveralfold by 0.005% to 0.05% digitonin, and approximatelyhalf of the enzyme activity then becomes sedimentable at1 1,000g. True enzyme precipitation, therefore, is occurringunder these conditions, and digitonin at 0.02% was henceforthused for the product-entrapment protocol.

Decreasing the concentration of Ca2' and Mg2+ in reactionmixtures from 2 mM and 5 mm to 1 mm and 0.5 mM,

686 WU ET AL.




100

0-

f._

.u

(n

80

60

40

20

n,u.00 0.10 0.20 0.30 0.40

CHAPS (%)

Figure 1. Effect of CHAPS concentration on CS activity. Assayswere conducted with 10 pL of solubilized enzyme as described in"Materials and Methods." CHAPS concentrations include the 0.06%CHAPS carried over from the solubilization procedure. Symbols: (0)digitonin held constant at 0.01%; (O) CHAPS-digitonin ratio (w/w)held constant at 6.0.

respectively, reduced nonspecific aggregation from approxi-mately 10 to 20% to negligible levels (not shown). At thelower levels of cations, the rate of callose synthesis is reducedby approximately 75%, but sufficient callose is still made in30 min to entrap the enzyme (see below).

Release ofCS activity from P1 required resuspension in thepresence of CHAPS and EDTA, with 0.1 to 0.2% CHAPSand 3 mm EDTA giving the best overall yield of enzymeactivity and the highest resultant specific activities (notshown). Increasing CHAPS beyond 0.2% led to significantlevels of enzyme inactivation.

80

0

0.

a

4

FigurEntral"Matethe alwere(P1) upellet

0

0XCD . o_j Q

. OL

-

.--

-0

a._c

U1

0 20 40 60 80

Incubation Time (min)

Figure 3. Time-course of glucan formation and product-dependentenzyme precipitation. A, Percentage of incorporation of added UDP-GIc into ethanol-insoluble glucan in a reaction mixture containing 1mM UDP-[14C]Glc, 0.01% digitonin, 1 mm CaCI2, 0.5 mm MgCI2, and0.22% CHAPS together with cellobiose and buffer. "Glucose Incor-poration" refers to the percentage of added [14C]Glc from UDP-[14C]Glc incorporated into ethanol-insoluble polymer. B, Time course ofprecipitation of CS into P1 following centrifugation at 7000g for 40min under the same conditions as A. Symbols: (0) full reaction mixtureas above incubated at 300C and (U) on ice; (0) same reaction mixtureminus UDP-Glc incubated at 300C and (0) on ice. "Activity in Pellet"refers to the total activity of the first pellet (P1) relative to the totalamount of solubilized activity initially present.

Correlation of Enzyme Sedimentation with ProductSynthesis

60 \ At the lower centrifugation speed, with altered concentra-

tions of reaction components, a time-dependent (Fig. 3) andUDP-Glc-dependent (Fig. 4) sedimentation ofCS is observed.

40 J The time course (Fig. 3) shows a correlation between product

formation (Fig. 3A) and precipitation of enzyme in P1 (Fig.3B). The control experiments (Fig. 3B), incubating the en-

zyme under a variety of conditions in which callose synthesis20 / \does not occur, showed no precipitation of activity. Further-

more, Figure 4 shows that precipitation is substrate-depend--0£ b - - o .ent. At 2 mm UDP-Glc, 55% of the activity sedimented (Fig.

u.00 0.01 0.02 0.03 0.04 0.05 0.06 4). At 5 mm, 64% sedimented (not shown in Fig. 4). Carry-over of unlabeled UDP-Glc from entrapment mixtures to

Digitonin (%) assay mixtures did not pose a problem in these experiments

because the sum of sedimented activity plus activity remain-e 2. Effect of digitonin and UDP-Glc on CS sedimentation.

pment reactions were conducted for 40 min as described in ing in the supernatant was not appreciably affected as UDP--rials and Methods" with varying digitonin concentrations and in Glc levels were increased from 0 to 5 mM. Furthermore, UDP-bsence (0) and presence (0) of 1 m UDP-Glc. The samples Glc was the only nucleotide diphosphate sugar that couldthen centrifuged at 11,000g and the activity of the first pellet cause sedimentation of the enzyme, with no sedimentation of

vas assayed. "Activity in Pellet" refers to the total activity of the activity or visible callose formation being observed with 1 mMrelative to the total amount of solubilized activity initially present. ADP-Glc, CDP-Glc, GDP-Glc, UDP-Gal, UDP-galacturonic

@* 0.01% Digitonin-- [CHAPSJ/[Dig.]=6

f W * |

687




14 {} Supernatant

X |* PelletC 120

a.b100

o 80

G0

= 40

> 20

0< 0 1000 2000

UDP-Glc (glM)

Figure 4. UDP-Glc-dependent sedimentation of CS. Reactions were

conducted for 40 min at 300C as described in Figure 3 followed bycentrifugation at 7000g for 40 min and assay of the supernatant andresuspended pellet. Symbols: (@) percentage of initial activity re-

covered in the first pellet (P1); (E) percentage of initial activity re-

covered in the first supernatant (S1).

acid, UDP-glucuronic acid, UDP-mannose, or UDP-N-acetyl-glucosamine. Taken together, these experiments show thattrue product-dependent sedimentation (product entrapment)is now occurring.

Table II shows that the specific activity of CS isolated byproduct-dependent sedimentation is improved by a factor ofthreefold relative to CHAPS-solubilized enzyme with yieldsof 40 to 50%. Specific activity was not further improvedfollowing release of the enzyme from the glucan. Controlsamples also shown in Table II illustrate that only 12% of the

CS activity added to the entrapment mixture sedimented at7000g in the absence of UDP-Glc and product synthesis, andonly 4% sedimented in the complete absence of divalentcations.

Purified enzyme (S2) from red beet showed unusually goodstability, with 50% retention of activity following 1 week ofstorage at 4°C. This could be due to small amounts of callosestabilizing the enzyme.

Combination of Product Entrapment with OtherEnrichment Steps. Glycerol Gradient Centrifugation

CS purified by product entrapment (S2 fraction) was furtherfractionated by rate-zonal centrifugation (160,000g for 1 h)on glycerol gradients containing either chelators or divalentcations (Fig. 5). In the presence of chelators, 81% of therecovered activity (76% of the total activity added) sedimentedas a peak between fractions 1 and 4, whereas with Ca2" andMg2+, 56% of the recovered activity was found in the pellet.These data clearly demonstrate that CS prepared in the pres-

ence of chelators and taken through the two-step CHAPS-solubilization procedure (cations, then chelators) followed byproduct entrapment (presence of cations) and released bychelators still remains susceptible to cation-inducedaggregation.

Glycerol gradient centrifugation did not result in significantenhancements in specific activity, with the distribution ofprotein following the same pattern as activity profiles (notshown). Moreover, peak polypeptide profiles were similar tothose in the S2 fraction loaded onto the gradient. This resultdiffers from the distribution of protein and activity observedon gradient centrifugation of protein solubilized in digitoninfrom red beet (8), mung bean, or cotton (12) membranes, butnot subjected to product entrapment, in which the CS activitysedimented ahead ofthe solubilized protein. Factors influenc-

Table II. Enrichment of CS by Product Entrapment: Effect of Divalent Cations and SubstrateProduct entrapment was conducted in reaction mixtures of 1.0 mL (0.076 mg protein) as described

in "Materials and Methods," centrifuging the product at 7000g for 40 min. The standard incubationmixture contained 0.2 mm EDTA, 0.2 mm EGTA, 1.5% glycerol, 5 mm cellobiose, 0.02% digitonin,0.12% CHAPS in 50 mM Tris/HCI, pH 7.5, with addition of cations and UDP-Glc shown below. Numbersin parentheses indicate enzyme yield after the second centrifugation, relative to the activity of the firstpellet (P1).

Fractionand Incubation ConditionsTotal Total Specific Purification Yield of

Activity Protein Activity Activityunits mg units mg-1 -fold %

Solubilized Enzyme 34.2 0.0760 449 1 100No AdditionsS1 51.9 0.0657 789 1.8 152P1 1.4 0.0043 319 0.7 4

+ 1 mM CaCI2, 0.5 mM MgC12S1 37.9 0.0622 606 1.3 111P1 4.1 0.0068 598 1.3 12

+ 1 mM CaCI2, 0.5 mm MgCI2, 1mM UDP-Glc

S1 20.1 0.0565 356 0.8 59P1 16.9 0.0126 1343 3.0 50S2 14.7 0.0126 1164 2.6 43 (840/)P2 0.8 0.0037 228 0.5 2 (27%)

688 WU ET AL.




c00coL._

a

E

0Ea

I._

._

4Fraction

Figure 5. Glycerol gradient centrifugation of CS following productentrapment and enzyme release. CHAPS-solubilized CS (0.72 mL)was enriched by product entrapment, released from the callose pellet,and centrifuged as described in "Materials and Methods." Gradientscontained either 1 mm CaCl2 and 5 mm MgCI2 (I), or 3 mm EDTAand 3 mm EGTA (0), in 0.1% CHAPS, 50 mm Tris/HCI, pH 7.5.Recoveries of activity in this experiment were 56% (with cations) and76% (with chelators). P, pellet; 8, top of gradient.

S 1 2 3 4 5 6 7 8 9 10 11 S. - -WLF- "

r71 -4

4~ ~ ~ 5

* _IX- 2

4t5 jlll! ll:-4

31 , It-iII."

Figure 6. Polypeptide profiles of CHAPS-solubilized and product-entrapped fractions (double-stained). Lane S, molecular mass stand-ards. Lane 1, microsomal membranes. Lane 2, PEG layer (PM) frompartitioned membranes. Lane 3, dextran layer. Lane 4, PM fromsucrose step-gradient. Lane 5, CHAPS-solubilized CS (single-stepsolubilization) from aqueous partitioning. Lane 6, CHAPS-solubilizedCS (two-step solubilization) from aqueous partitioning. Lane 7,CHAPS-solubilized CS from sucrose gradient. Lane 8, "cationicallyprecipitated" CS from partitioned PM. Lane 9, "product-entrapped"CS from partitioned PM. Lane 10, "cationically precipitated" CS fromsucrose step-gradient. Lane 11, "product-entrapped" CS from su-crose step-gradient. Each lane contained 5 ,g of protein.

Table Ill. Product Entrapment of PM-Enriched Fractions Preparedby Gradient Centrifugation and Two-Phase Partitioning

Product-entrapped samples were prepared by incubation with 1mM Ca2+, 1 mm UDP-Glc, at either low (0.5 mM) or high (5 mM) levelsof Mg2+, and other effectors as described in Table II, and werecentrifuged at 7000g for 40 min.

Specific Activity Yield of ActivityFraction

Gradient Two-phase Gradient Two-phase

units mg-1 (-told) %Microsomal membranes 47 43 100 100Plasma membranes 165 (4) 768 (18) 74 28Solubilized 518 (11) 749 (17) 60 16EntrappedLow Mg2+ 1962 (42) 1311 (30) 35 5High Mg2+ 1795 (38) 1515 (35) 55 16

ing the aggregation state of polypeptides comprising the prod-uct-entrapped complex warrant further investigation.

Product Entrapment of PM-Enriched FractionsPrepared by Aqueous Two-Phase Partitioning

Once entrapment parameters were determined using gra-dient-purified PM, the product-entrapment procedure wasapplied to PM-enriched fractions prepared by two-phase par-titioning. This procedure has the advantage of producinghighly purified PM preparations (27). However, for conduct-ing large-scale characterization experiments such as the onesdescribed above, two-phase partitioning is more laboriousbecause yields of purified PM are significantly lower (0.0022versus 0.024 mg PM protein g-' tissue). Table III comparesthe two procedures. Microsomal CS activity was enriched 18-fold by aqueous partitioning as opposed to 3.5-fold by sucrosegradient centrifugation, but activity yields were less than half(28 versus 74%). Beyond the PM isolation step, a greater than35-fold enrichment of specific activity relative to microsomalmembranes was obtained, but yields ofPM produced by two-phase partitioning ranged only between 5 and 16% (comparedwith 55% in gradient-purified PM).Two additional characteristics of the PM prepared by

aqueous partitioning were highly significant. First, entrappedfractions from these PM contained fewer contaminating poly-peptides than did entrapped fractions from gradient-purifiedPM, and second, these PMs were free of the 55-kD ATP-binding subunit of the tonoplast ATPase (see below).

Polypeptide Composition of the Product-EntrappedEnzyme. Electrophoresis Profiles

Figure 6 shows double-stained polypeptide profiles of start-ing material and product-entrapped fractions obtained fromPM prepared by sucrose-gradient centrifugation and aqueoustwo-phase partitioning. This gel also distinguishes polypeptidepatterns obtained by time- and product-dependent "entrap-ment" and product-independent "cationic precipitation."Analysis of this gel and others show that numerous polypep-tides were removed by the solubilization and entrapmentprocedures, but that at least a dozen significant polypeptides

689




still remained in the product-entrapped preparations made by"entrapment" (Fig. 6, lanes 9 and 1 1).The major polypeptides or groupings of polypeptides com-

mon to each of the "entrapped" or "cationically precipitated"samples (Fig. 6, lanes 8-1 1) fell into the following ranges ofapparent molecular size (based upon the molecular masses ofstandards indicated at the sides of each gel): 92, 83, 70, 67,57, 43 (a broad band), 35, 31/29 (typically a broad band),and 27 kD (also a broad band). Polypeptides of >100 and 36kD are probably the most significant ones removed fromsoluble preparations by the entrapment procedure (Fig. 6,compare lanes 5-7 with 8-1 1). Various similarities betweenpolypeptide profiles of these entrapped preparations, andpolypeptides visualized from other tissue sources, are outlinedin the "Discussion."

Several important differences in polypeptide staining pat-terns pertaining to the use of two-phase partitioning must benoted. Most significant is the removal of polypeptides atmolecular masses of 67, 54, and 52 kD (compare lane 2 withlane 4). These polypeptides, which are invariably present inproduct-entrapped preparations made from gradient-purifiedPM, partition into the dextran layer during two-phase parti-tioning (lane 3) and represent subunits of the tonoplast ATP-ase that are carried over during the gradient PM purificationprocedure (see below). The aqueous two-phase method alsoremoved a large number of low molecular mass polypeptidesbetween 10- and 25-kD (compare lanes 2 and 4, and lanes 3and 6 with 7).

Further Characterization of Polypeptides in the57- to 52-kD Region

Based upon photolabeling results (1 1), we expected to ob-tain significant enrichment of one or more polypeptides inthe 57-kD region in product-entrapped preparations. Thevarious fractions in Figure 6 show at least four polypeptidesin the range of 57 to 52 kD that could overlap with theposition of the photolabeled 57-kD polypeptide of Frost et al.(1 1). Two of these polypeptides (54 and 52 kD) were elimi-nated by two-phase partitioning. Because the ATP-bindingsubunit of the tonoplast ATPase is a 55-kD polypeptide (19),we hypothesized that these subunits (at 54 and 52 kD in ourgel system) might derive from tonoplast membranes contam-inating the gradient PM preparation. This was confirmedimmunologically. We excised the polypeptides at 54 and 52kD and generated polyclonal antibodies. These antibodies(Fig. 7) reacted with the 54- and 52-kD polypeptides in allfractions derived from gradient-purified PM (Fig. 7, lanes 2,4, and 6) but did not react with any of the fractions derivedfrom PM prepared by partitioning (Fig. 7, lanes 1, 3, and 5).The antibody directed against the 54-kD polypeptide alsoreacted with the 55-kD subunit of purified tonoplast ATPase(Fig. 7, lane 7). Conversely, antibodies directed against the55-kD (19) subunit of the tonoplast ATPase cross-reactedwith the 54-kD polypeptide found in gradient-purified PM(not shown). As expected, neither immunoinhibition norimmunoprecipitation of CS activity was obtained by theseantibodies.

w '4

54yv

5 2z 4'

Figure 7. Western blot of polyclonal antibodies raised against the54- and 52-kD polypeptides. Lane 1, PM from two-phase partitioning.Lane 2, PM from sucrose step-gradient. Lane 3, CHAPS-solubilizedCS (two-step solubilization) from aqueous partitioning. Lane 4,CHAPS-solubilized CS from sucrose gradient. Lane 5, "product-entrapped" CS from partitioned PM. Lane 6, "product-entrapped" CSfrom sucrose step-gradient. Lane 7, Purified tonoplast ATPase fromref. 16. Each lane contained 5 lg of protein.

DISCUSSION

Product entrapment is a straightforward method for enrich-ment of large amounts of solubilized CS and other glycosyltransferases in a relatively short period compared with otherpurification steps such as gel filtration chromatography (23),glycerol gradient centrifugation of digitonin-solubilized en-zyme (3-6, 12), or HPLC of CHAPS-solubilized enzyme (10,21). It has generally been assumed that synthesis of polysac-charide is necessary to achieve enzyme enrichment by thisprocedure, but the relevant mechanistic aspects were notaddressed in previous studies (3, 10, 12, 15).

Initial attempts to enrich CHAPS-solubilized CS by productentrapment were conducted under the assumption that puri-fication was occurring predominantly by attachment ofCS toits glucan product, or by entrapment of the enzyme within ameshwork of glucan ("product entrapment"). Thus, the find-ing that similar yields of CS, and increases of specific activity,could be obtained independent of glucan formation (Table I)was unexpected. This phenomenon is due to nonspecificprecipitation of the enzyme as the combined result of adecrease in detergent levels and an increase in divalent cationconcentration. It has already been shown that divalent cationsinhibit CS solubilization (8, 23) and that the enzyme solubi-lized with digitonin sediments faster in glycerol gradientscontaining cations than it does in the presence of chelators(12). Here we show that CS solubilized in CHAPS and en-riched by product entrapment is also prone to cation-inducedaggregation (Fig. 5). In accordance, Delmer et al. (4) demon-strated that, for detergent-solubilized proteins ofcotton fibers,certain combinations of cations induce precipitation of CSactivity and a specific set of polypeptides. True product en-trapment (Table II) proceeds with lower yield because onlyhalf the enzyme activity is precipitated.

Enrichments in CS activity were generally in the order of

690 WU ET AL.

.!




three- to fourfold, whether the procedure followed was cationprecipitation and high-speed centrifugation (Table I) or trueproduct entrapment and low-speed centrifugation (Table II).In comparison with previous reports ofisolation ofchitin (13)and cellulose (15) synthase by product entrapment, yields inthis system are relatively high (40-50%), but the degree ofpurification is significantly lower. This is probably due tononspecific precipitation of other proteins occurring evenunder our modified conditions. Nearly identical results wereobtained with CHAPS-solubilized CS from celery, except thatthe enzyme was somewhat more unstable during the resus-pension step (not shown). Our results contrast with those ofHayashi et al. (12), who reported a 50- to 200-fold increasein specific activity.Active CS is readily dissociated from the callosic-product

matrix by EDTA and high levels of CHAPS, and addition ofpreformed glucan to solubilized enzyme does not cause itsinsolubilization (our unpublished results). The nature of theinteraction between CS and its reaction product is unclear,but cannot be very strong.During the course of this study it became apparent that

callose synthesis in most preparations is strongly inhibited at0.6% CHAPS, the concentration used for solubilization (Fig.1). Because activity is restored when the CHAPS levels arediluted to 0.12% or less following solubilization in the pres-ence of chelators, the ability of the enzyme to catalyze calloseformation may be directly dependent on reversible changesin the physical state of the enzyme protein complex and thephospholipid that surrounds it. A phospholipid requirementfor CS is well established (23, 24, 26). Divalent cations atmillimolar levels not only activate CS and change the solu-bility in alkali of the glucan product derived from mung bean(12), but also strongly influence the aggregation state of theenzyme. In addition, divalent cations inhibit solubilization ofmany proteins from the membrane and can precipitate manyof the proteins dissolved from membranes in the presence ofchelators (23).

In the photolabeling study, cation-induced product-entrap-

ment was a key tool for implicating a 57-kD UDP-Glc bindingpolypeptide as the UDP-Glc-binding subunit of CS (11).Closer analysis of double-stained polypeptide profiles (Fig. 6)show that a 57-kD polypeptide is present. However, in frac-tions derived from PM prepared by centrifugation on sucrosestep gradients, two intensely staining polypeptides at 54 and52 kD are also evident (Fig. 6, lanes 10 and 11). Thesepolypeptides, which are removed when PM is isolated byaqueous two-phase partitioning (Fig. 6, lanes 9 and 10), arecomponents of the tonoplast ATPase (19) carried over duringsucrose gradient centrifugation (Fig. 7). Although CS yieldsare significantly compromised by two-phase partitioning(Table III), resultant electrophoresis profiles (Fig. 6) confirmthat the partitioning step represents an essential step in thepreparation of high-purity CS fractions (9, 10, 22).

Polypeptide profiles from a wide range of tissue sourceshave now been reported (Table IV), and it is apparent thatmany similarities are emerging. Polypeptides in the 58 to 48kD range have now been documented in enriched CS prepa-rations from five plant and one fungal species (Table IV). Thefact that several other polypeptides or groups of polypeptidesseen here (Fig. 5), such as the ones at 70, 43, 35, 31/29, and27 kD, have been observed in partially pure CS preparationsfrom other sources, raises speculation concerning a commonsubunit structure consisting of integral and peripheral mem-brane proteins.

Also of interest is that this preparation and one from cotton(4, 5) contain polypeptides in the 83 to 85 kD range, whichare of similar molecular size range as the UDP-Glc-bindingpolypeptide of cellulose synthase from Acetobacter xylinum(16). A 42-kD polypeptide has been reported to bind UDP-pyridoxal in mung bean (21). The polypeptides in the 35 to32 kD range found here and in purified fractions from fourother sources (Table IV) could be proteolyis products of alarger polypeptide (4). We also observe several smaller sizedbut prominently staining polypeptides at 31/29 and 27 kD.Both of these photolabel with 5-azido-uridine 5'-f3-diphos-phate glucose (1 1), and polypeptides in this size range corre-

Table IV. Similarities in Polypeptide Composition Between Enriched Callose and Cellulose SynthasePreparations from Various SourcesAsterisks indicate polypeptides observed to incorporate UDP-Glc-containing photolabels.

Tissue Source and Reference

11e17 Brassica Gossypium Pisum Apium Glycine Saprolegnia Acetobacter

thisstudy) (10) (4,5) (6,7) (22) (9) (3) (15,16)Molecular mass estimated mass (k)

range (kD)93-83 92 90 93

83 84 83 83*72-65 70 66 66 72

67 6558-48 57* 57 58 55 56 50

52* 4846-41 43 46 4135-32 35 35 34 35 34

32 3231-28 31/29 28 29 30 31 3028-25 27 25 26 28 25

691




late with activity profiles in glycerol gradients of cotton (5)and pea (6). These may bear similarity to a 3 1-kD polypeptidereported by Fink etal. (9) in purified preparations of soybeanCS. At this point, it cannot be concluded that any of thesepolypeptides are components of the CS complex, but the factthat similar polypeptides are enriched in CS preparations fromdiverse sources, using a variety of enrichment methods, pro-

vides a foundation upon which to build a structural model. Itwill be interesting to see how these data are reconciled.

In summary, regardless of the mechanism by which purifi-cation is achieved (by true entrapment or by cation-inducedaggregation), the precipitation and resuspension of CHAPS-solubilized CS represents a rapid and effective enrichmentprocedure. Additional steps to further improve the purity ofthe CS complex and to identify its polypeptide componentsare under investigation.

Note Added in Proof

Affinity photolabeling of a 3 1-kD polypeptide in a purified glucansynthase preparation from a grass has recently been reported (MaiklePJ, Nj K, Hoogenraad NJ, JohnsonE, Stone BA [1991] Immunopre-cipitation and photoaffinity labeling of a UDP-Glc binding polypep-tide of a (3-glucan synthase from Lotium multiflorum. Abstracts, 15thInternational Congress of Biochemistry, Jerusalem, Israel, August 4-8, 1991, p 39).

ACKNOWLEDGMENTS

We wish to thank Dr. Philip Rea of the University of Pennsylvaniafor the gift of tonoplast ATPase and antibodies directed against it.The assistance of Mrs. Pamela McPartland with manuscript prepa-

ration is deeply appreciated.

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25. Wasserman BP, Frost DJ, Lawson SG, Mason TL, Rodis P,Sabin RD, Sloan ME (1989) Biosynthesis of cell wall polysac-charides: membrane isolation, in vitro glycosyl transferase assayand enzyme solubilization. In H-F Linskens, JF Jackson, eds,Modern Methods of Plant Analysis, New Series, Vol 10. Sprin-ger-Verlag, Heidelberg-New York, pp 1-11

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