Plant Physiol. (1988) 88, 172-1770032-0889/88/88/0172/06/$0 1.00/0

Purification and Properties of Glucoamylase from Sugar BeetCells in Suspension Culture

Received for publication March 1, 1988 and in revised form April 3, 1988

HIROSHI MASUDA*, MANABU MURATA, TOSHIMASA TAKAHASHI, AND SHIRO SUGAWARADepartment ofAgricultural Chemistry, Obihiro University ofAgriculture and Veterinary Medicine,Obihiro, Hokkaido 080, Japan

ABSTRACT

Glucoamylase and a-amylase are present in callus and suspensioncultures of sugar beets (Beta vulgaris L.) as well as in mature roots. Thesubcellular localization of glucoamylase differed in callus and suspension-cultured cells: in callus, glucoamylase was present together with a-amylase in the soluble fraction of cells, but in suspension cultures, it waspresent predominantly in the extracellular fraction while most of the a-amylase activity remained in cells. Glucoamylase activity was consider-ably lower in callus protoplasts relative to the activities of a-mannosidaseand a-galactosidase and the suspension of callus in Murashige-Skoogliquid medium or in mannitol by brief agitation resulted in the release ofglucoamylase to the medium. These findings suggest that glucoamylasein callus may be present in a soluble form in the free space in the cellwall. Both mature roots and callus contained a-amylase and glucoamylasein the soluble fraction. Glucoamylases in the soluble fraction of callusand in the medium of suspension cultures were purified separately tohomogeneity by the same four-step purification procedure, which includedfractionation with ammonium sulfate, column chromatography on car-boxymethyl cellulose, gel filtration on Bio-Gel P-150, and preparativedisc electrophoresis. The identity of the glucoamylases from the twosources was confirmed by a comparison of chromatographic behaviorduring purification, mobility during gel electrophoresis, Mr (83,000 D bySDS PAGE), and enzymic and kinetic properties of the catalytic reaction,such as optimal pH and temperature, heat stability, and K, value forsoluble starch. Glucoamylase from suspension cultures was one of themajor proteins that were secreted into the medium. Dedifferentiation ofleaves of young plants to callus was accompanied by induction of gluco-amylase and repression ofsome a-amylases and the debranching enzyme.

Enzymes that hydrolyze starch, such as glucoamylase, a-am-ylase, and the debranching enzyme, are found in mature rootsof sugar beet (1 1). These roots store large amounts of sucrose butnot starch. The occurrence, the subcellular localization, and thepossible physiological functions of glucoamylase and a-amylasein callus and suspension-cultured cells have been examined.Suspension cultures provide an attractive system for a variety ofbiochemical studies so it is important to compare such cells withcallus and plant tissues.

Cells of higher plants in suspension culture secrete variousenzymes into the medium (1, 2, 4, 6, 7, 10, 12). However, thephysiological roles of extracellular enzymes in higher plants stillremain unclear. A useful initial approach to this problem is tocompare the localization of the enzymes in suspension-culturedcells with that in callus and intact organs.We found a-amylase and glucoamylase in mature roots and

in both callus and suspension-cultured cells. Glucoamylase in

callus and suspension-cultured cells showed different patterns oflocalization: it was present in the soluble fraction of callus cellsbut mostly in the medium of suspension cultures.

MATERIALS AND METHODS

Plant Materials. Sugar beet (Beta vulgaris L.) plants weregrown on vermiculite for 4 weeks in a growth chamber, under a16 h/8 h day-night cycle at 24C.Cell Culture. Sugar beet calluses were generated on Murashige-

Skoog (MS) salts agar medium (14), supplemented with 0.25mg/L BA, from the leaves of young plants by the methoddescribed by Saunders and Daub (16) and were subculturedevery 3 weeks on MS agar medium without plant hormones.Suspension cultures of cells were initiated by transferring callusinto MS liquid medium without plant hormones after 7 d, sievingthrough nylon screen with 295 um pores, collecting on nylonscreen with 85 um pores, and incubating on a reciprocal shakerin the light at 27°C. Suspension-cultured cells were subculturedevery 7 d in MS liquid medium without plant hormones.

Purification of Glucoamylase from Suspension Cultures. Me-dium (3.2 L) was separated from 12-d-old cultured cells (520 gfresh weight) by vacuum filtration, and solid ammonium sulfatewas added to a final concentration of 80% saturation. Aftercentrifugation, the precipitate was dissolved in a small amountof 5 mm Na-acetate buffer (pH 5.6) that contained 5 mM ,B-mercaptoethanol and was dialyzed overnight against the samebuffer. After centrifugation of the dialyzed solution, the super-natant was applied to a CM-cellulose column (2.5 x 30 cm)preequilibrated with 5 mm Na-acetate buffer (pH 5.6) that con-tained 5 mm /3-mercaptoethanol. The column was washed withthe same buffer to remove unadsorbed proteins and was elutedwith a linear gradient from 0 to 0.5 M NaCl in 500 ml of thebuffer. The active fractions were pooled and concentrated byprecipitation with ammonium sulfate at 80% saturation. Aftercentrifugation, the precipitate was dissolved in a small amountof 10 mm phosphate buffer (pH 7.4) that contained 5 mM ,3-mercaptoethanol and 0.1 M NaCl and then was applied to a Bio-Gel P-150 column (5 x 70 cm) preequilibrated with the samebuffer. Proteins were eluted with the same buffer. Active fractionswere pooled, concentrated in vacuo to about 0.5 ml in a Coblo-dion-bag, and applied to a running column for preparative discelectrophoresis, pH 4.3. The equipment consisted of a runningcolumn (1.0 x 15 cm) and two chambers. The running columnwas composed of 10 mL of a separating gel and 0.8 mL of astacking gel. The gels and electrode buffer were prepared by themethod described by Reisfeld et al. (15), using pH 4.3 gel (15%)and /3-alanine acetate buffer (pH 4.5). Sucrose and a smallamount of f3-alanine acetate buffer (pH 4.5) were added to thesolution of enzyme, and the mixture was layered on the stackinggel. Electrophoresis was carried out at 5 mamp at 4°C. After 16h, the gel was transferred to a solution of I0- M 4-methylumbel-

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GLUCOAMYLASE IN SUSPENSION-CULTURED CELLS

liferyl-a-D-glucoside (pH 4.4), and the enzyme was localized byUV fluorescence. The site of the enzymic activity was cut outand the protein was recovered electrophoretically from the gelusing an Extraphor Electrophoretic Concentrator (Mitsumi,Tokyo).

Purification of Soluble Glucoamylase from Callus. Three-week-old callus (230 g fresh weight) was homogenized with threevolumes of 10 mm Na-phosphate buffer (pH 7.4) that contained5 mM f3-mercaptoethanol in a Polytron (Kinematica) for 3 min.The homogenate was filtered through a layer of 42 gm nylonscreen. Subsequent purification procedures, including ammo-nium sulfate precipitation, chromatography on CM-cellulose andBio-Gel P- 50, and preparative electrophoresis, were the sameas those for purification from suspension cultures.

Preparation of Wall-Bound Enzyme. The cell wall fraction,obtained by filtering the homogenates of cultured cells, waswashed thoroughly with distilled water and suspended in 0.1%(w/v) sodium deoxycholate. After 2 h, the cell walls were washedthoroughly with distilled water to remove deoxycholate. Thesepurified cell walls were used as the preparation of wall-boundenzyme.

Isolation of Protoplasts. Callus was incubated in digestionmedium, which consisted of 1% Cellulase "Onozuka" RS (Yakult

0 3 6 9 12 15Time ( day )

FIG. 1. Changes in cell growth and activities of a-amylase and glu-coamylase in soluble and extracellular fractions of sugar beet suspensioncultures. Values of growth given represent the means of triplicate deter-minations, and the vertical bars indicate the SD. (a) soluble fraction; (b)extracellular fraction. (0) a-amylase; (0) glucoamylase.

Table I. Localization ofa-Amylase and Glucoamylase in Callus andSuspension CulturesCallus Suspension Culture

Enzyme Soluble Cell wall Soluble Cell wallfraction fraction fraction fraction ediu

total unitsa-Amylase 35.2 6.2 49.0 8.1 0Glucoamylase 52.3 7.1 16.0 6.2 92.3

E

CD-I.n

._

-U-0

.)co

ENaw

C

%._

co0

(U

coL,a)

C0C.)

cz

10 20 30 40 50 60Fraction number (20 ml/tube)

FIG. 2. Column chromatography on DEAE-cellulose of a-amylaseand glucoamylase in the soluble fraction of callus and in soluble andextracellular fractions of suspension-cultured cells. Soluble fractions (a)of callus, and soluble fraction (b) and extracellular fraction (c) of suspen-sion-cultured cells. (0) a-amylase; (0) glucoamylase.

Co., Ltd., Tokyo Japan), Pectolyase Y-23 (Seishin Co., Ltd.,Tokyo Japan), 2% Driselase (Kyowa Hakko Kogyo Co., Ltd.,Tokyo Japan) and 0.7 M mannitol (pH 5.8), at 27°C for 4 h.EDTA was added to the suspension at a final concentration of 2mM. After 10 min, the suspension was centrifuged at 1,000 rpmfor 5 min in a swinging bucket rotor. The protoplasts floated tothe upper surface and were pipetted into 0.7 M mannitol andthen washed by repeated centrifugation at 1,000 rpm for 5 minwith 0.7 M mannitol until none of the washings contained a-mannosidase, a-galactosidase, or amylase activities. The finalprotoplasts (1 mL volume from 23 g callus) were used for analysisof glycosidase activities.

Preparation ofAmylase from Young Leaves and Mature Roots.Four-week-old leaves, of the same age as those from which thecallus was derived, were used. Mature roots were harvested atthe Experimental Farm at Obihiro University. The leaves werehomogenized with three volumes of 10 mm phosphate buffer(pH 7.4) that contained 5 mM ,3-mercaptoethanol. The homog-enate was squeezed through a layer of cotton gauze, solid am-monium sulfate was added to 80% saturation, and then thesuspension was centrifuged at 12,000g for 20 min. The fractionthat precipitated at 80% saturation was dissolved in a smallamount of 5 mm phosphate buffer (pH 7.4) that contained 5 mMfl-mercaptoethanol, and the solution was dialyzed overnightagainst the same buffer. After centrifugation of the dialyzedsolution, the supernatant was applied to a DEAE-cellulose col-umn (2.5 x 30 cm), preequilibrated with 5 mm phosphate buffer

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Plant Physiol. Vol. 88, 1988

FIG. 3. Protoplasts of sugar beet callus (magnification, x375).

(pH 7.4) that contained 5 mm ,B-mercaptoethanol. The columnwas washed with the same buffer to remove unadsorbed protein,and proteins were then eluted with a linear gradient from 0 to0.5 M NaCl in 500 mL ofbuffer. Mature roots were homogenizedwith three volumes of 10 mm phosphate buffer that contained0.5% (w/v) sodium isoascorbate in a Polytron. Subsequent pro-cedures were the same with those used for preparation ofamylasefrom leaves.Assay of Amylase and Debranching Enzyme. The reaction

mixture consisted of 0.25% (w/v) soluble starch or pullulan, 20mM citrate buffer (pH 4.4 for acid amylase, pH 6.8 for neutralamylase, and pH 5.6 for pullulanase) and 300 ML of enzymesolution in a final volume of 1 mL. The mixture was incubatedat 37°C for 30 min. The amount of reducing sugar liberated wasdetermined by the method ofSomogyi-Nelson (17). The amountof reducing sugar was calculated by subtracting the backgroundobtained when the enzyme solution containing j-mercaptoeth-anol was used. One unit of activity of acid (glucoamylase) orneutral amylase (a-amylase) was defined as the amount of en-zyme which catalyzed the production of 1 ,mol ofreducing sugar

as glucose or maltose per min at 37°C, repectively. One unit ofdebranching enzyme activity was defined as the amount ofenzyme that catalyzed the production of 1 ,umol of reducingsugar as maltotriose per min at 37TC.

Protein Estimation. Protein concentration was estimated byBradford's method, using BSA as a standard (3).Paper Chromatography. Descending paper chromatography

was performed using a solvent system of 1-butanol:pyr-idine:water (6:4:3). Reducing sugars on paper were visualized bythe method of Trevelyan et al. (19).Gel Electrophoresis. PAGE was performed by the method of

Reisfeld et aL (15) using a 15% gel at pH 4.3. The buffer usedwas ,B-alanine acetate (pH 4.5). Electrophoresis was carried outfor 120 min at 4 mamp/tube. Proteins were stained with Coo-massie brilliant blue G-250, and carbohydrates with periodicacid-Schiff (8).SDS-PAGE of Proteins from Culture Medium. Three volumes

of ethanol were added to the medium after removal of cells. Theprecipitate was suspended directly in 500 ,l ofstandard gel buffer,and undissolved material that remained after boiling was re-moved by centrifugation. An aliquot (150 ,l) of the supernatantwas used as a sample for SDS-PAGE. SDS-PAGE was performedon 10 to 15% (w/v) polyacrylamide gradient gels using the buffersystem ofLaemmli (9). Proteins were visualized by staining withCoomassie brilliant blue R.

RESULTS AND DISCUSSIONEvidence That the Amylases of Callus Were Glucoamylase

(Acid) and a-Amylase (Neutral). Callus derived from leaf ex-plants of a young plant, and also the mature roots, containedacid and neutral amylases, as defined by the optimum pH oftheir respective catalytic activities. They were well-separated bychromatography on DEAE-cellulose after fractionation of crudeextracts of callus with ammonium sulfate. Acid amylase pro-duced glucose as the sole product from soluble starch, amylopec-tin, i3-limit dextrin, and rabbit liver glycogen (data not shown).No oligosaccharides were liberated from soluble starch through-out the course of the reaction. Thus, the acid amylase was aglucoamylase. By contrast, neutral amylase liberated maltose asthe main product and small amounts ofglucose and oligosaccha-rides from the a-glucans, a reaction characteristic of a-amylase.These acid and neutral amylases were apparently the same asthose of roots of mature sugar beets (1 1).

Localization of Glucoamylase and a-Amylase in SuspensionCultures and in Callus. The changes in activities of a-amylaseand glucoamylase during growth ofsuspension culture are shownin Figure 1. A logarithmic rate of growth was observed between3 and 9 d after subculture. In the soluble fraction, a-amylaseactivity remained high until the 12th d, i.e. until early stationaryphase, and then the activity disappeared. Levels of glucoamylaseremained low throughout growth. Glucoamylase activity in themedium increased steadily throughout the logarithmic and sta-

Table II. Distribution ofGlucoamylase, a-Mannosidase, and a-Galactosidase in Whole Extracts, ProtoplastsofCallus, and the Medium

As described in "Results and Discussion," callus was transferred into MS liquid medium and agitated for90 min. Glycosidase activities were measured in the medium obtained by the filtration of the suspension weremeasured.

Enzyme Whole Callus Extracts Protoplasts MediumTotal units

Glucoamylase 84.3 0.70 60a-Mannosidase 2.6 0.28 0.08a-Galactosidase 2.9 0.36 0.12Glucoamylase/a-mannosidase 32.4 2.5 750Glucoamylase/a-galactosidase 29.1 1.9 500

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GLUCOAMYLASE IN SUSPENSION-CULTURED CELLS

Table III. Purification ofGlucoamylasefrom Suspension Cuftures and Callus ofSugar Beet

Culture Volume Total Protein Total activity Specific activity Purification Recovery

mL

Secretory glucoamylase from suspension cultiCrude extracts 3200 180% (NH4)2SO4 200CM-cellulose 220Bio-Gel P-150 50Preparative electrophoresis 1

Intracellular glucoamylase from callusCrude extract 750 880% (NH4)2SO4 110 4CM-cellulose 180Bio-Gel P-150 60Preparative electrophoresis 1

C

units/mg pro-mg units tein

1184106059450592

60841522714230

6.715.130.076.5102.2

0.71.07.2

72.894.7

MW

94

67

43

30

20.1

14.1FIG. 4. Disc gel electrophoresis ofglucoamylase in the soluble fraction

of callus and in the extracellular fraction of suspension-cultured cells. C,callus; S, suspension culture. Electrophoresis was carried out as describedin "Materials and Methods."

tionary phases of growth, but a-amylase activity was barelydetectable. Thus, in suspension-cultured cells, a-amylase wasmostly present predominantly in the soluble fraction, whileglucoamylase was found predominantly in the medium. Thesoluble fraction from callus contained both glucoamylase and a-

amylase (Table I), but only traces ofthese amylases were detectedin the cell wall fractions of both suspension-cultured cells andcallus. Glucoamylase eluted in the unadsorbed fraction, and a-

amylase eluted in the adsorbed fractions during chromatographyon DEAE cellulose (Fig. 2). In suspension-cultured cells, gluco-amylase activity from cells was much lower in the soluble fractionthan a-amylase activity. Most of the glucoamylase activity wasfound in the medium (Fig. 2). These results suggest that the

FIG. 5. SDS-PAGE of purified glucoamylase and proteins from themedium. The Mr standards were: a-lactalbumin (14.4 kD), soybeantrypsin (20.1 kD), carbonic anhydrase (30 kD), ovalbumin (43 kD), BSA(67 kD), and phosphorylase (94 kD); lane 1, purified glucoamylase fromsuspension-culture; lane 2, purified glucoamylase from callus; lane 3,proteins in the medium. Electrophoresis was carried out as described in"Materials and Methods."

soluble fraction of callus contained glucoamylase, most ofwhichwas released upon exposure to liquid medium.We attempted to distinguish between the intracellular and

extracellular localization of glucoamylase in callus by preparingprotoplasts (Fig.3 ) and assaying the activity in protoplasts andextracts of callus (Table II). The activities of a-mannosidase anda-galactosidase, which are vacuolar enzymes and are only se-

2.34.511.415.3

1.39.7

98.4128.0

1009050438

1006837235

2 31

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Plant Physiol. Vol. 88, 1988

0

EI-O

%._

.r_

C.)coC:

0

I ..O

a

o.2._"co-

0a0C00

z

10 20 30 40 50 60Fraction number (20ml/tube)

FIG. 6. Chromatography on DEAE-ellulose of amylases in the solu-ble fraction of mature roots and leaves. (a) Mature roots: (0) pH 4.4activity (glucoamylase); (0) pH 6.8 activity (a-amylase). (b) Leaves: (0)pH 4.4 activity; (0) pH 6.8 activity.

creted at low levels into the medium during suspension culture,were also measured. Their high levels of activity indicated thatthe protoplasts were both intact and viable. The ratio of gluco-amylase to a-mannosidase and to a-galactosidase activitieschanged from 32.4 and 29.1, respectively, in extract of wholecallus, to 2.5 and 1.4 in protoplasts. This result suggests thatglucoamylase may be an extracellular enzyme in the callus.Glucoamylase is considered to be localized in the cell wall

(a) (b)XIoo

~8O60

> 40

0 20

0

compartment as either the free form or the bound form. Thepresence of the enzyme in soluble form in the free space of thecallus cell wall is indicated by the results of the following exper-iments. Callus was transferred into MS liquid medium andagitated for 90 min. As shown in Table II, most of the gluco-amylase was released from callus into the medium after briefagitation, whereas only traces of a-mannosidase and a-galacto-sidase were detected in the medium. This result suggests thatmost of the glucoamylase, which was confined in soluble formin the free space ofthe callus cell wall, could be released into themedium when the callus was dissociated into single cells or smallclumps by agitation in liquid medium. This release of gluco-amylase is also observed when MS liquid medium is replaced by0.7 M mannitol. It is probable that glucoamylase is present almostexclusively in soluble form in the extracellular space ofthe callus,since bound enzymes cannot generally be released from cell wallsby mannitol, even though the association with the cell wall isonly loose. This localization of glucoamylase may also apply tointact tissues, such as mature roots.The polysaccharides in hypocotyls ofpeas have been extracted

from the extracellular matrix by centrifugation methods (18).The linamarin ,3-glucosidase in leaf discs of Costa Rican limabeans was found, by an analysis of protoplasts and cell walls (5),to be confined to the apoplast. These results suggest that the freespace of the cell wall may contain a variety of macromolecules,such as proteins and polysaccharides. In the suspension culturesof sugar beet cells, when the cells were cultured in MS liquidmedium prepared with pH values between 3 and 8.5, the pH ofall cultures returned to pH 5.4 to 5.6 (our unpublished data).The cell walls, like the vacuoles, are well known to be acidic. Itis, thus, possible that the free space in cell walls of callus, whichconsists of an amorphous mass of multilayered tissues, may bean environment similar to the medium of suspension culturesand may sequester materials such as polysaccharides and proteinswhich are secreted from suspension-cultured cells.The enzymes that were present in the soluble fraction in the

homogenate of intact tissues and callus have been considered sofar to be intracellular enzymes. However, if some portion of the

2 3 4 5 6 7 8 2 345 6 78 2 3 456 7 8pH

FIG. 7. Effect ofpH on activities from callus (a), leaves (b), and mature roots (c). In callus and mature roots, (0): glucoamylase; (0): a-amylase.In leaves, (0): peak I; (0): peak II; (A): peak III (peaks as shown in Fig. 6).

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GLUCOAMYLASE IN SUSPENSION-CULTURED CELLS

enzymes is present in a soluble form in the extracellular space,the location of soluble enzymes in intracellular and extracellularfractions should be reinvestigated.Glucoamylases in Both Callus and Suspension-Cultured Cells

Are the Same Enzyme. Glucoamylases from the two sourceswere purified separately to homogeneity, using the same proce-dure, which included the four steps described in "Materials andMethods." Summaries ofthe purifications are presented in TableIII. Glucoamylase from callus was purified approximately 120-fold with a recovery of 5%, and the enzyme from suspension-cultured cells was purified approximately 15-fold, with 8% re-covery. Both purified enzymes moved toward the cathode essen-tially as a single band on disc electrophoresis, and their mobilitieswere the same (Fig. 4). The same values of optimal pH, namely,pH 4.4; of optimal temperature for a 20-min reaction, namely,70C; of heat stability after preincubation for 10 min at 65C;and ofKm for soluble starch, namely, 7.8 mg/ml, were obtainedwith each preparation. Each single band was also stained byperiodic acid-Schiff reagent, suggesting that the enzymes areglycoproteins (data not shown). SDS-PAGE showed that bothenzymes have Mr of 83,000 D (Fig. 5). Thus, the glucoamylasein the soluble fraction of callus and the glucoamylase in themedium of suspension cultures can be purified by the sameprocedures and are probably the same enzyme.Glucoamylase was a predominant extracellular protein in the

suspension cell medium, as is apparent from the analysis ofSDS-PAGE (Fig. 5). However, the enzyme had only low activity inthe cell wall fraction. This difference suggests the direct secretionof the enzyme into the medium. One of the two extracellularacid invertases behaves similarly (12).

Soluble amylases in Leaves and Mature Roots of Sugar Beets.The chromatographic behavior on DEAE-cellulose of solubleamylases in the leaves of young sugar beets from which calluswas derived and of soluble amylases in the mature roots areshown in Figure 6. Soluble amylases in leaves were eluted asthree peaks (peaks I, II, and III). Each peak contained a-amylase,as judged by criteria described previously. The enzymes in ma-ture roots were separated into glucoamylase and a-amylase, andthe elution profiles were in good agreement with those of theenzymes in callus (1 1).The effect of pH on the activities of the three amylases from

leaves and the two from mature roots was determined betweenpH 3.0 and 8.0 (McIlvaine's buffer) (13) (Fig. 7). Glucoamylaseand a-amylase from mature roots had identical pH-activityprofiles to those of the enzymes isolated from callus. One (peakIII) of the three a-amylases from leaves was eluted in approxi-mately the same fraction as the a-amylases from callus andmature roots, but the other two amylases (peaks I and II) werequite different.The debranching enzyme was present both in leaves (0.126

unit/g fresh weight) and in mature roots (0.028 unit/g freshweight) but was hardly detectable in callus and suspension-cultured cells.The leaves ofyoung plants contained at least three isoenzymes

ofa-amylases and one debranching enzyme but no glucoamylase,

while in callus, initiated from the leaves, a glucoamylase wasnewly synthesized, but two of the a-amylases and the debranch-ing enzyme disappeared. This observation suggests that the ini-tiation of callus from intact tissue was accompanied by theinduction ofglucoamylase and the repression ofsome a-amylasesand the debranching enzyme. The induction ofalkaline invertasealso accompanied the initiation of suspension cultures of cellsfrom leaf explants of young plants (12). Thus, these resultsprovide evidence that the "dedifferentiation" of intact tissuescaused significant changes in the expression of several enzymicactivities.

Glucoamylase, a glycoprotein and a predominant extra-cellular protein, may be a useful protein through which to studythe mechanism of extracellular secretion of enzymes andglycoproteins.

LITERATURE CTED

1. ASAMIZU T, Y INOUE, A NISHI 1981 Glycosidases in carrot cells in suspensionculture: Localization and activity change during growth. Plant Cell Physiol22: 469-478

2. BLIGNY R, R DOUCE 1983 Excretion of laccase by sycamore (Acer pseudopla-tanus L.) celis. Purification and properties of the enzyme. Biochem J 209:489-496

3. BRADFORD MM 1976 A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle for protein-binding.Anal Biochem 72: 248

4. CHAUBET N, A PAREILLEUX 1982 Characterization of jS-galactosidase ofMed-icago sativa suspension-cultured cells growing on lactose. Effect ofthe growthsubstrates on the activities. Z Pflanzenphysiol 106: 401-407

5. FREHNER M, EE CoNN 1987 The linamarin j3-glucosidase in Costa Rican wildlima beans (Phaseolus lunatus L.) is apoplastic. Plant Physiol 84: 1296-1300

6. GASPAR T, C KEVERS, C PENOEL, H GREPPIN 1983 Auxin control of calcium-mediated peroxidase secretion by auxin-dependent and auxin-independentsugar beet cells. Phytochemistry 22: 2657-2660

7. HISAJIMA S, T ITO 1983 Activity and cellular distribution of disaccharides incultured cells of Japanese morning-glory. Agric Biol Chem 47: 107-109

8. KONAT G, H OFFNER, J MELLAH 1984 Improved sensitivity for detection andquantitation of glycoproteins on polyacrylamide gels. Experientia 40: 303-304

9. LAEMMLI U K 1970 Cleavage of structural proteins during assembly of thehead of bacteriophage T4. Nature 227: 680-685

10. MASUDA H, Y OZEKI, S AMINO, A KOMAMINE 1985 Changes in the activitiesof various glycosidases during carrot cell elongation in a 2,4-D-free medium.Plant Cell Physiol 26: 995-1001

11. MASUDA H, T TAKAHASHI, S SUGAWARA 1987 Purification and properties ofstarch hydrolyzing enzymes in mature roots of sugar beets. Plant Physiol 84:361-365

12. MASUDA H, T TAKAHASHI, S SUGAWARA 1988 Acid and alkaline invertases insuspension cultured cells of sugar beet. Plant Physiol (in press).

13. MCILvAINE TC 1921 A buffer solution for colorimetric comparison. J BiolChem 49: 183-186

14. MURASHIGE T, F SKooG 1962 A revised medium for rapid growth and bioassayswith tobacco tissue cultures. Physiol Plant 15: 473-497

15. REISFELD RA, UT LEwIs, DE WILLIAMS 1962 Disk electrophoresis of basicproteins and peptides on polyacrylamide gels. Nature 195: 281-283

16. SAUNDERS JW, ME DAUB 1984 Shoot regeneration from hormoneautonomouscallus from shoot cultures of several sugar beet (Beta vulgaris L.) genotypes.Plant Sci Lett 34: 219-223

17. SOMOGYI M 1952 Notes on sugar determination. J Biol Chem 195: 1918. TERRY ME, BA BONNER 1980 An examination of centrifugation as a method

of extracting an extracellular solution from peas, and its use for the study ofindoleacetic acid-induced growth. Plant Physiol 66: 321-325

19. TREVELYAN WE, DP PRocrOR, JS HARRISON 1950 Determination of sugarson paper chromatograms. Nature 166: 444445

177

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