Plant Physiol. (1986) 81, 913-9180032-0889/86/81/091 3/06$0 1.00/0

Gel-Electrophoretic Separation, Detection, and Characterizationof Plant and Bacterial UDP-Glucose Glucosyltransferases

Received for publication January 31, 1986 and in revised form April 4, 1986

MICHAEL P. THELEN' AND DEBORAH P. DELMER*ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, California 94568

ABSTRACI

We have developed procedures for detection and characterization ofUDP-glucose: glucosyltransferases following electrophoretic separationin nondenaturing polyacrylamide gels. Using digitonin-solubilized mem-brane protein preparations from a variety of plants and two cellulose-producing bacteria, activity can be demonstrated for several UDP-glu-cose:6-glucan synthases with an in situ assay following gel electropho-resis. These enzymes can be characterized within the gels with respectto effector requirements and products produced, and several advantagesof this assay over solution assays are demonstrated. For example, theclear dependence of plant UDP-glucose:(1-.3)-fi-glucan synthase on bothCal2 and a j-linked glucoside is shown; bacterial cellulose synthasesshow direct stimulation within the gel by guanyl oligonucleotide, and theAcetobacter xylinum enzyme appears more stable in the gel assay thanin solution assay.

Plants and microorganisms produce a variety of polysaccha-rides which are secreted to the cell wall or extracellular medium.However, particularly in plants, little is known about the prop-erties of isolated enzymes which function in the biosynthesis ofsuch polysaccharides. One major reason for this lack of infor-mation is that most, if not all, of these enzymes are associatedwith cellular membranes, and attempts to purify polysaccharidesynthases from membranes by conventional methods have metwith little success. It is also difficult to detect distinct, but minor,activities in crude preparations containing other more abundantsynthases. Although several investigators have reported solubili-zation of membrane-associated synthase activities using nonde-naturing detergents such as digitonin (1, 5-7, 10, 1 1), the indi-vidual proteins responsible for activity have not been identified.Among the many methods for separation of proteins, electro-

phoresis in polyacrylamide gels probably has the highest resolvingpower, especially when used with SDS or other denaturants. Insitu assays for various enzymes in nondenaturing polyacrylamidegels have been reported (8), but only recently have we succeededin detecting a native glucan synthase by this method (5). Kanget al. (12) have also recently used a native-gel assay to detectdigitonin-solubilized chitin synthase. In this report, we demon-strate that several UDP-glucose:,i-glucan synthases of plant andbacterial origin can be detected and characterized after separationby nondenaturing PAGE, and we demonstrate some of theadvantages of this assay over solution assays.

' Present address: Biochemistry Department, Tennis Court Road,University of Cambridge, Cambridge, CB2 lQW, England.

MATERIALS AND METHODS

Enzyme Preparation. Agrobacterium tumefaciens (LBA 4404,a gift of B. Simpson, ARCO Plant Cell Research Institute) wasgrown in LB broth on a gyrotory shaker at 28°C. Acetobacterxylinum (a gift of C. Weinhaus and M. Benziman ofthe HebrewUniversity, Jerusalem, Israel) was cultured according to Aloni etal. (1). Digitonin-solubilized membrane proteins that includedcellulose synthase activity were prepared from A. xylinum andA. tumefaciens as described (1), except that bacteria were dis-rupted in a French pressure cell at 20,000 p.s.i. instead of bysonication. Stem tissue from 10 g (fresh weight) of etiolated pea,mung bean, or soybean seedlings (4-7 d old) was homogenizedusing a mortar and pestle at 4°C in 10 ml of 50 mm Tris-Cl, pH7.5, containing 5 mM EGTA and 5 mM EDTA, and filteredthrough 2 layers of Miracloth, and centrifuged 5 min at 10OOgto remove cell walls. Membranes were sedimented from thesupernatant at 100,000g for 60 min, resuspended in 1 ml of 50mM Tris-Cl (pH 7.5), containing 20% glycerol, and stored at-80°C. Digitonin-solubilized proteins were obtained by resus-pending isolated membranes to their original volume in 125 mMTris-Cl (pH 6.8), containing 10% glycerol and 1% digitonin2(Serva) and then incubating for 20 min on ice, followed bycentrifugation at 100,000g for 60 min. Supernatants were care-fully removed and stored at -80°C.

Polyacrylamide Gel Electrophoresis. Samples were applieddirectly as the digitonin supernatants; after application ofsamples(5-10 gl each containing 10-50 gg protein), electrophoresis wascarried out using the discontinuous buffer system of Laemmli(16) without the addition of SDS. A Hoeffer vertical mini-slabgel apparatus was used; the small size of the gels (0.75 mm x 8cm x 7 cm) reduces the volume of solution required in theactivity assay and enhances diffusion ofsubstances (e.g. substrate,effectors) between gel and solution. Stacking gels were 4.5%acrylamide and separating gel concentrations are described inthe figure legends. Native-gel electrophoresis was performed at4°C, at 10 to 15 mamp per gel.

Native-Gel Assay. Following electrophoresis, each gel was cutinto sections containing 1 or 2 sample lanes, and rinsed in 100ml of 10 mm Tris-Cl (pH 7.5) (or 20 mM Na-Hepes [pH 7.3]with similar results) for 30 min, with one change of buffer after15 min. Each gel section was then placed in a plastic Petri dishand incubated for enzyme activity at room temperature in 50mM Tris-Cl (pH 7.5) (or 20 mm Na-Hepes [pH 7.3] with similarresults), 3 mm NaN3, UDP-['4C]-glucose (ICN Radiochemicals),and cofactors as described in the text. Unreacted radiolabeledsubstrate was removed from gel sections by rinsing twice for 20min each in 100 ml of 40% ethanol, 5 mm EDTA, 10 mM Tris

I We obtained 1% digitonin by dilution from an aqueous solution of100 mg digitonin/ml after heating in a boiling water bath for severalminutes and removing any remaining insoluble material by centrifuga-tion or filtration.

913 www.plantphysiol.orgon January 21, 2020 - Published by Downloaded from

Copyright © 1986 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

THELEN AND DELMER

(pH 7.5), followed by successive 20 min washes in 100 ml of 10mM Tris-CI (pH 7.5) until the radioactivity in the wash bufferwas reduced to background. To detect radiolabeled product, gelswere treated with either PPO (New England Nuclear) in aceticacid (21) or with 'Amplify' (Amersham), dried and exposed tofilm (Kodak X-omat AR) at -80°C for 18 to 36 h.An alternative method was employed to detect highly active

glucan synthases. Following electrophoresis, the gel sections wererinsed in buffer as above, incubated in 50 mM Tris-Cl (pH 7.5),3 mM NaN3, 5 mm UDP-glucose (unlabeled), and cofactors asdescribed in Figure 1. The (3-glucan product in the gel wasvisualized by its fluorescence under UV light after staining in 50ml of 0.1% Cellufluor (Polysciences) for 30 min, and destainingfor 2 h in 100 ml of buffer.

Quantification of Activity. Bands containing radiolabeledproduct (as detected by fluorography) were excised from thedried gel, placed in scintillation fluid (Beta-max, Westchem), andcounted in a liquid scintillation spectrometer. Unlabeled sectionsof equal size were counted for background radioactivity. Noattempts other than visual have been made to quantify productdetected by fluorescent staining of product with Cellufluor.

Product Analyses. After incubating with substrate and washingto remove unincorporated radioactivity, gel sections were placedin 5 ml of 50 mm sodium acetate (pH 5.5), containing 3 mMNaN3 and one of the following: no additions (control); 2 mg/mllaminarinase (Mollusca, CalBiochem); 0.2 mg/ml cellulase(Streptomyces partially purified enzyme preparation, a gift of E.T. Reese; U.S. Army Laboratory, Natick, MA); or 50 mm sodiummetaperiodate. The enzyme digestions were carried out for 24 hat 37°C. Periodate oxidation took place over 5 d at 4°C in thedark according to the method of Hay et al. (9). The periodate-treated gel was further processed at room temperature as follows:several rinses in buffer; overnight incubation in 0.5 M NaBH4;several extensive washes in water; 8 to 12 h incubation in 0.5 MHCI; several rinses in water. Gel slices treated with protease K(Merck) at 100 ,ug/ml in 10 mm Tris-Cl (pH 7.3) were incubated18 h at room temperature. Control slices in buffer lackingprotease were incubated similarly. After enzyme or periodatetreatments, gel sections were processed for staining or fluorog-raphy and quantitated as described above.

RESULTS

Glucan Synthase Activities Can Be Detected in a Native-GelAssay. Of several nondenaturing detergents tested for ability tosolubilize plant glucan synthase activity (CHAPS, Zwittergents,cholate, Triton, and digitonin), digitonin was found to give bestsolubilization of activity as judged either by solution assay ofactivity or by the gel assay described below; for example, usingmung bean membranes containing 5 mg protein per ml, weobtained optimal solubilization of (l- 3)-(3-glucan synthase ac-tivity using a 20 min extraction at 4°C in a buffered solution of1% (w/v) digitonin. Under these conditions, about 30% of themembrane-associated activity and about 40% of total proteinwas solubilized. CHAPS allowed some limited solubilization, butZwittergent and Triton X-100 were completely ineffective. Dig-itonin has also been found to be most effective for solubilizationof the A. xylinum cellulose synthase (1) and was used in thisstudy for the bacterial enzymes analyzed.

Following separation by nondenaturing PAGE, these solubi-lized activities can be analyzed by incubating the washed gel withappropriate substrate and effectors, subsequent removal of un-used substrate by washing, followed by use of a suitablevproductdetection procedure. The gel in Figure 1 demonstrates such anassay for a digitonin-solubilized preparation derived from etio-lated pea seedling membranes. Sections cut from the gel wereincubated for various time intervals in a solution containingUDP-glucose and two effectors, celloboise and Ca2", recently

reported to be required for plant membrane-associated (l-3)-(3-glucan synthase activity (4, 5, 13, 14). After incubation, thesections were stained with Cellufluor, a 4,4'-diaminostilbene-type fluorescent brightener which binds specifically to ,3-linkedglucans (18); the increase in fluorescence intensity of the bandduring the time course of incubation should therefore be a directresult of the catalytic formation of ,B-linked glucan within thegel. The fact that intensity increases with time of incubationindicates that the staining is not due to the preexisting glucan inthe preparations. Insolubility of the ,B-glucan produced helps toimmobilize it in the gel matrix, thus allowing prolonged incu-bation (in this case up to 42 h) and staining without substantialloss of product.

Following an 18 h incubation in assay solution, section 5 wastreated with a solution of laminarinase, a (1--3)-fl-glucanasepreparation which is incapable of digesting (1--4)-(3-glucan (THayashi, DP Delmer, unpublished data). Such treatment resultedin nearly complete elimination of fluorescence and thus suggeststhat this product is (l-+3)-(-glucan. We have found that thisglucanase will digest the product even after it is complexed withCellufluor (data not shown).

Polysaccharide synthases can also be detected by fluorographyof a gel in which radiolabeled product has been formed, giving amore sensitive and quantitative assay than the staining proce-dure. Section 1 of Figure 2A shows activity detection of the samepea glucan synthase as that of Figure 1, designated here as bandA. This section was incubated for 18 h in UDP-['4C]glucose,Ca2", cellobiose, and buffer as before; following fluorography,the regions containing radioactivity were excised and counted.Under these conditions, 1800 pmol ofglucose were incorporatedinto insoluble polymer by this enzyme. Bands containing as littleas 100 cpm (0.2 pmol) of glucose incorporated into product canbe detected after exposing the film for 18 h.From these results it is clear that this glucan synthase from

peas maintains activity for an extended time within a polyacryl-amide gel, and can be identified by the detection and character-ization of its product. A more detailed study of the time courseof reaction for this enzyme using an extract from etiolated mungbean seedlings indicates that product formation in the gel islinear with respect to time for about 2 h and continues to increaseat a slower rate for many hours thereafter. In a similar experi-ment, we compared initial rates of activity for the mung beanenzyme in a gel assay to that in a solution assay of similarcomposition and found the activities to be virtually identical.

Individual Activities Are Distinguished by Their Mobility andEffector Requirements. In a search for other polysaccharidebiosynthetic enzymes, we tested the effects ofdifferent incubationconditions in the native-gel assay. Using the pea preparation, anadditional minor and diffuse band of radioactivity designatedband B, was detected in section 2 (Fig. 2A) when the specificactivity of the UDP-['4C]glucose was increased. Removing cel-loboise from the assay eliminated the activity ofthe major glucansynthase A (section 3 Fig. 2A), but did not affect appearance ofband B. In another experiment using a mung bean preparation(Fig. 2B), we studied the effector requirements for these twoactivities in more detail using high specific activity substrate.Band A clearly requires a combination of Ca2" and cellobiosefor optimal activity; band B is also Ca2" dependent, but does notrequire cellobiose. Trace amounts of Ca2" present in the experi-ment shown in Figure 2A, section 3, were apparently sufficientto allow activity since a combination of Mg2e and EGTA are noteffective (Fig. 2B, sections 3 and 4). Bands A and B have alsobeen detected under the same conditions of those in Figure 2A,section 2, for peas, using preparations derived from developingcotton fibers, etiolated soybean seedlings, and barley coleoptiles.

In addition to the plant enzymes, a preparation of A. tumefa-ciens, which produces cellulose in vivo (17), gives a band of

914 Plant Physiol. Vol. 81, 1986

www.plantphysiol.orgon January 21, 2020 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

UDP-GLUCOSE GLUCOSYLTRANSFERASES DETECTED IN GELS

SECTION I 2 3 4 5

E

TIME (h) 0.5 1.5 18 42 18OF ASSAYFIG. 1. Activity detection of a solubilized pea glucan synthase activity with a fluorescent dye. Membranes prepared from stem tissue of dark-

grown pea seedlings were treated with digitonin; replicate samples containing 10 Mg of solubilized proteins (l00,OOOg digitonin-supernatant) wereseparated by electrophoresis in a 7.5% acrylamide gel. Each one-lane section cut from the gel was incubated in 2 ml of 50 mm Tris Cl (pH 7.5), 5mM UDP-glucose, 5 mm CaC12, and 10 mm cellobiose for the time intervals indicated. Buffer-insoluble product formed was visualized by staininggel sections with Cellufluor; after destaining, the gel was illuminated with UV light for photography. Section 5 was incubated as above for 18 h, andfurther incubated in laminarinase prior to staining (see "Materials and Methods" for complete details). Arrows on the left indicate the top andbottom of the separating gel, and the direction of enzyme migration during electrophoresis is shown on the right.

product when incubated in 10 jsM UDP-['4C]glucose and Mg2",as shown in section 4 of Figure 2A. An activity band of similarmobility was observed under the same incubation conditionsfrom a preparation containing the cellulose synthase (1) of A.xylinum (see product analysis section below). Evidence that theactivities detected using A. xylinum or A. tumefaciens are truecellulose syntheses comes from our observation that these activ-ities within the gels are stimulated by addition of the guanyloligonucleotide known to activate the A. xylinum enzyme systemin vitro (20). For the A. xylinum enzyme, stimulation by 4 sMg/ml ofguanyl oligonucleotide ranged in various experiments from10- to 40-fold over controls; for A. tumefaciens, stimulation wasless pronounced (approximately 4-fold). None of the bacterial orplant activities described here was affected in the gels by 100 gM2,6-dichlorobenzonitrile, a specific inhibitor ofcellulose synthesisin vivo in plants.The proteins responsible for the radioactive band B from plants

and the bands from the bacterial preparations clearly migratewell within the separating gels; however, the major band A fromthe plant preparations focuses at the interface between the 4.5%acrylamide stacking gel and a 7.5% separating gel (Fig. 1), and itbarely enters a 6% separating gel (Fig. 2). Such low mobility isconsistent with our observations (MP Thelen, DP Delmer, un-

published data) that the (1-+3)-fl-glucan synthase, solubilized bydigitonin from mung bean or soybean membranes, has a mol wtgreater than half a million daltons as judged by gel filtration orsedimentation in glycerol gradients; it is unknown, however, howmuch the native size may be modified by association withdigitonin or endogenous lipid. This enzyme, as solubilized, runsas a distinct peak in 15 to 30% (v/v) glycerol gradients centrifugedat 100,000g for 20 h; therefore, the activity does not appear tobe still associated with membrane fragments of any substantialsize. Conditions which will allow further migration ofthe enzymeinto the separating gel are shown in section 5 of Figure 2A forthe cotton fiber enzyme; migration of a rather diffuse band ofactivity well into the gel can be observed in a 4.5 to 10%acrylamide gradient.

Individual Activities Are Further Deflned by Product Analyses.With the discovery of electrophoretically distinct activity bandsthat differ in effector requirements, we used several techniquesto analyze the nature ofthe radioactive products formed by theseproteins. Because the products remain immobilized, their struc-ture can be at least partially characterized in the gel followingthe standard assay. Table I shows the results of four complemen-tary treatments that were used to analyze the products generatedby plant and bacterial preparations: (a) digestion with laminari-

915

www.plantphysiol.orgon January 21, 2020 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

THELEN AND DELMER

A4 B.3tEC; floNband

_ *; 4

At Co2A~~~~~~Co A

B

A

B

I

A 90650 40600 40 Ndcpmr/band -At 790

B1 50 380L530A' .... .r--:-

AB, ;;~.

. Am I ...a

-o...I- --

..

FIG. 2. Detection and characterization of glucan synthase activities by radiolabeled product formation. A, Digitonin-solubilized membraneproteins (10 Ag each) from pea seedling, sections I to 3, and A. tumefaciens, section 4, were separated by electrophoresis in a 6% acrylamide gel.Cotton fiber proteins were used in section 5 and the separating gel contained a 4.5 to 10% gradient of acrylamide. Each section of the gel wasincubated separately for 18 h in 2 ml of 50 mm Tris-Cl (pH 7.5), with substrate and cofactors as listed below, then processed for fluorography andquantitated as described in "Materials and Methods." The fluorogram shown was exposed to film for 36 h. Section 1 and 5: 100 AM UDP-['4C]glucose (50 cpm/pmol), 10 mm cellobiose, 5 mM CaCI2; section 2, 10 AM UDP-['4C]glucose (500 cpm/pmol), 10 mM cellobiose, 5 mm CaCI2; sections3 and 4, 10 AM UDP-['4C]glucose (500 cpm/pmol), 5 mM MgCl2. Activity bands from pea samples (A and B) and A. tumefaciens (At) are labeledon the left, cotton (cot A) on the right, and quantitative values for each band are given in cpm below the gel. ND = not determined. B, Similar gelas in A, using digitonin solubilized proteins from mung beans (22 Ag per lane). Incubation time for assay was 2 h; all assays contained 5.4 AM UDP-['4C]glucose (544 cpm/pmol), 20 mM Na-Hepes (pH 7.3), and the effectors each at 5 mm as indicated. Section 1, EDTA; section 2, CaCI2; section 3,MgCl2 and EGTA; section 4, MgCI2, EGTA, and cellobiose; section 5, EGTA and cellobiose; section 6, CaCl2 and cellobiose; section 7, CaCl2,MgC92, and cellobiose.

Table I. Analyses ofProducts Generated by Enzymes in Native-Gel AssayPercent of Product' Remaining in Gel after Treatment

Enzyme Assayed Effector within Native Gel Requirements Smith

Laminarinase Cellulase degradation Protease K

Mung bean or soybean'Band A Ca2+/cellobiose 0.1-5 1-18 78-100 60-100cBand B Ca2` 76-90 25-32 2-16

UDP-GLUCOSE GLUCOSYLTRANSFERASES DETECTED IN GELS

ride matrix.The product synthesized by the A. xylinum preparation was

extensively degraded by cellulase or Smith degradation, but mostremained after laminarinase or protease treatment. This result,coupled with the observation that activity is stimulated by guanyloligonucleotide, supports the conclusion that this enzyme is thecellulose synthase characterized previously in both membrane-bound and solubilized forms by Aloni et al. (1) and Ross et al.(20).

Analyses of the plant Band B product gave surprising results.As for A. xylinum, the product was resistant to laminarinase, butdegraded by cellulase and the periodate procedure; however,unlike A. xylinum, the product of Band B was completely re-moved by treatment with Protease K. Thus, this band couldrepresent a UDP-glucose-binding protein or a glucoprotein gen-erated by attachment of a limited number of sugar residues to aspecific protein. Requirement for high specific activity substrateto detect this band would be consistent with either of theseinterpretations, as would the kinetics of its formation with radio-activity being incorporated rapidly for only the first 15 to 30 minof incubation.

DISCUSSIONIn this report, we demonstrate that several distinct UDP-

glucose: (3-glucan synthases can be detected and characterizedusing an in situ assay following solubilization and electrophoreticseparation in non-denaturing polyacrylamide gels. The mostactive of these was the Ca2+-cellobiose-dependent (l-)3)-fi-glu-can synthase of plant origin. To date, this activity has beendetected using this assay from every plant tissue we have exam-ined, representing examples from dicotyledonous (mung bean,soybean, and pea seedlings and cotton fibers) and monocotyle-donous (barley) plants. The absolute dependence of this enzymeactivity for both Ca2' and a ,B-glucoside such as cellobiose is mostclearly seen in the native-gel assay. From these results and otherrecent studies with solution assays of this enzyme by our group(4, 5) and that of Kauss (13, 14), it is clear that the old criteriaestablished by Ray (19) for detection of glucan synthase II(plasma membrane-localized (I-3)-/3-glucan synthase assayedwith high substrate concentration and no divalent cation) needto be reevaluated. In extracts without chelators, endogenous Ca2+levels are usually sufficient to saturate this enzyme, and cellobiosemay not be required if the preparations are not washed or containsucrose or glycerol which at high concentrations can substitutefor cellobiose (DP Delmer, unpublished data). We have alsofound that in the presence of Ca2' and cellobiose, the Km forUDP-glucose is 0.2 to 0.3 mM; if Ca2" is removed the Km isshifted to above 1 mM. The enzyme therefore can be detectedwithout divalent cation in crude preparations at high substrateconcentration, and Ray's initial criteria for assay were acceptable,but clearly not optimal. Thus, the gel-assay has been useful to usfor clarifying the true effector requirements of this enzyme.

In most of these plant preparations, we were also able todistinguish an electrophoretically distinct and far less prominentCa2"-dependent activity, the so-called band B activity. At present,the nature of this activity remains obscure since the low levels ofradioactivity generated have hampered further detailed charac-terization of the radioactive material. The Ca2" dependence ofthis activity suggests a relationship to (1--3)-f,-glucan synthesis,but the product is resistant to laminarinase treatment and issolubilized instead by protease treatment indicating that it isprobably associated with a polypeptide rather than being highmol wt j3-glucan. This activity could represent a glucan synthasewhich initiates limited polymerization such as a protein primeror a glucoprotein intermediate; alternatively it might represent aUDP-glucose binding protein.

activity in these assays similar to the well-characterized Golgi-localized enzyme described by Ray (19). Assay conditions shownin Figure 2B, sections 3 or 4 (high specific activity UDP-glucose,MgCl2 ± cellobiose) should have been optimal for detection ofthis activity; the minor band of low mobility in section 4 issusceptible to laminarinase (data not shown) and so probablyrepresents partial substitution by Mg2e of the Ca2' requirementfor (1--3)-,B-glucan synthase. It may be that the Golgi-localizedenzyme is poorly solubilized by digitonin and/or unstable underthe separation and assay conditions.

Cellulose synthase activities from two bacteria, A. xylinumand A. tumefaciens, have also been successfully demonstrated. Astudy of these activities has demonstrated several useful advan-tages of the native-gel assay over solution assays. One advantagehas been the ability to demonstrate that a known activator ofthein vitro A. xylinum enzyme system, guanyl oligonucleotide (20),acts by directly interacting with the enzyme itself. We also wereable to demonstrate some activation of the A. tumefaciens en-zyme in the gel assay even though we have not been able to doso reproducibly in solution assays (J Cooper, DP Delmer, un-published data). Thus, in the gel assay enzymes may be separatedrelatively easily from potential inhibitors which exist in crudeextracts. Such separation may also explain our observation thatthe A. xylinum enzyme, which is extraordinarily unstable inextracts at ambient temperature, maintains at least some activityfor many hours in the gel assay; immobilization within the gelmatrix could also play a role in stabilization.There are, however, some disadvantages ofthe gel assay. Some

digitonin-solubilized membrane proteins tend to aggregate andnot all proteins appear to enter and be resolved in well in theseparating gel. For minor activities requiring high specific activitysubstrate, the cost ofthe assay using radioactive nucleotide sugarscan be rather high. We have sought to minimize these costs byusing minigels; if necessary, the unused substrate can be reiso-lated for further use. The small amounts of product generated inmost cases make it difficult to determine the precise structure ofproducts generated. The techniques for product analyses usedhere allowed us to identify with some certainty the plant (1- 3)-f,-glucan synthase and bacterial cellulose synthases, but cautionis advised and a number of different analyses are necessary forprecise identification of products. For example, in a preliminaryreport of this work (22) we tentatively concluded that the nativeband B is a (1--4)-fl-glucan synthase based on susceptibility ofthe product to cellulase and Smith degradation and resistance tolaminarinase; later studies showing digestion by Protease K leavesopen the question as to what is the structure of the productgenerated by this band.

Nevertheless, it is clear that the gel assays can be highlysensitive and can allow simultaneous detection of several distinctactivities; minor activities which may be masked in the presenceof more abundant synthases can be characterized in ways notusually possible in solution assays. In theory, enzymes catalyzingthe synthesis of any homopolymer (and ofsome heteropolymers,e.g. DNA, see Ref. 2) can be resolved and characterized by themethods described here. For example, water-soluble productscould be precipitated with an appropriate solvent in order tofacilitate their retention in gels for further analyses. Enzymeswhich modify polysaccharides or other polymers, e.g. by sulfationor acetylation, should be detectable if an appropriate substrate iscast into the gel prior to electrophoresis or diffused into the gelafterward. In addition to detection of products by radiolabeling,use of product-specific probes such as dyes, lectins (12), ormonoclonal antibodies should also be possible.An increasing number of enzymes are being successfully re-

natured following SDS-PAGE (2, 3, 15). We have attempted torenature the A. xylinum cellulose synthase from SDS-gels withoutsuccess. However, using SDS-solubilized proteins of plant origin,

917

We did not detect any obvious plant ( 1 --*4')-#-glucan synthase www.plantphysiol.orgon January 21, 2020 - Published by Downloaded from

Copyright © 1986 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

918 THELEN AND DELMER

we have detected production of a labeled band at 70 to 73 kDfollowing incubation of washed SDS gels in UDP-['4C]glucoseand MnC12. The nature of this activity is currenty under inves-tigation and will be the subject of a future communication.

Acknowledgments-We are especially grateful to J. Cooper for preparing Agro-bacterium tumefaciens samples, and thank him and our other colleagues, T. Hayashiand S. M. Read, for many helpful ideas. We extend our appreciation to C. Weinhausand M. Benziman for their generous gift of their strain ofAcetobacter xylinum andfor advice in preparation of guanyl oligonucleotide, and to E. T. Reese for thepreparation of cellulase. We also thank Karen Long for her skillful preparation ofthe manuscript.

LITERATURE CITED

1. ALONI Y, R COHEN, M BENZIMAN, D DELMER 1983 Solubilization ofthe UDP-glucose: 1,4-fl-D-glucan 4-f3-D-glucosyltransferase (cellulose synthase) fromAcetobacter xvlinuim. J Biol Chem 258: 4419-4423

2. BLANK A, JR SILBER, MP THELEN, CA DEKKER 1983 Detection of enzymaticactivities in sodium dodecyl sulfate-polyacrylamide gels: DNA polymerasesas model enzymes. Anal Biochem 135: 423-430

3. BLANK A, RH SUGIYAMA, CA DEKKER 1982 Activity staining of nucleolyticenzymes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis: useof aqueous isopropanol to remove detergent from gels. Anal Biochem 120:267-275

4. DELMER DP, M THELEN, M MARSDEN 1984 Regulatory mechanisms for thesynthesis of jB-glucans in plants. In WM Dugger, S Bartnicki-Garcia, eds,Structure, Function, and Biosynthesis of Plant Cell Walls. American Societyof Plant Physiologists, Washington, DC, pp 133-149

5. DELMER DP, G COOPER, D ALEXANDER, J COOPER, T HAYASHI, C NITSCHE,M THELEN 1985 New approaches to the study of cellulose biosynthesis. JCell Sci Suppl 2: 33-50

6. EIBERGER LL, CL VENTOLA, BP WASSERMAN 1985 Solubilization of a digi-tonin-stable glucan synthase from red beet root. Plant Sci Lett 37: 195-198

7. FLOWERS HM, KK BATRA, J KEMP, WZ HASSID 1968 Biosynthesis of cellulosein vitro from uridine diphosphate D-glucose with enzymic preparations fromPhaseoluis aureuts and Luipinus albuts. Plant Physiol 43: 1703-1709

Plant Physiol. Vol. 81, 19868. GABRIEL 0 1971 Locating enzymes on gels. Methods Enzymol 22: 578-6049. HAY GW, BA LEWIS, F SMITH 1963 Determination ofthe average chain length

of polysaccharides by periodate oxidation, reduction and analysis of thederived polyalcohol. Methods Carbohyd Chem 3: 367-370

10. HEINIGER U 1983 UDP-glucose: 1,3-,B-glucan synthase in potato tubers: solu-bilization and activation by lipids. Plant Sci Lett 32: 35-41

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

12. Kang MS, N Elango, E Mattia, J Au-Young, PW Robbins, E Cabib 1984Isolation of chitin synthetase from Saccharomyces cereviseae. J Biol Chem259: 14966-14972

13. KAuss H, H KOHLE, W JEBLICK 1983 Proteolytic activation and stimulationby Ca2" of glucan synthase from soybean cells. FEBS Lett 158: 84-88

14. KOHLE H, W JEBLICK, F POTEN,W BLASCHEK, H KAUSS 1985 Chitosan-elicitedcallose synthesis in soybean cells is a Ca2" dependent process. Plant Physiol77: 544-551

15. LACKS SA, SS SPRINGHORN 1980 Renaturation of enzymes after polyacryl-amide gel electrophoresis in the presence of sodium dodecyl sulfate. J BiolChem 255: 7467-7473

16. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

17. MATTHYSSE A, KV HOLMES, RHG GURLITZ 1981 Elaboration of cellulosefibrils by Agrobacterium tumefaciens during attachment to carrot cells. JBacteriol 145: 583-595

18. RATTEE ID, MM GREUR 1974 The Physical Chemistry of Dye Absorption.Academic Press, New York, pp 181-182

19. RAY PM 1979 Separation of maize coleoptile cellular membranes that beardifferent types of glucan synthetase activity. In E Reid, ed, Plant Organelles.Horwood Publishers, Chichester, UK, pp 135-146

20. Ross P, Y ALONI, C WEINHOUSE, D MICHAELI, P WEINBERGER-OHANA, RMEYER, M BENZIMAN 1985 An unusual guanyl oligonucleotide regulatescellulose synthesis in Acetobacter xylinum. FEBS Lett 196: 191-196

21. SKINNER MK, MD GRISWOLD 1983 Fluorographic detection of radioactivityin polyacrylamide gels with 2,5-diphenyloxazole in acetic acid and its com-parison with existing procedures. Biochem J 209: 281-284

22. THELEN M, D DELMER 1985 The use of gel electrophoresis for separation anddetection of native and renatured polysaccharide synthase activities. FedProc 44: 1408

www.plantphysiol.orgon January 21, 2020 - Published by Downloaded from Copyright © 1986 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org