Plant Physiol. (1987) 85, 131-1360032-0889/87/85/0131 /06/$0 1.00/0

Modulation of Pea Membrane J-Glucan Synthase Activity byCalcium, Polycation, Endogenous Protease, and ProteaseInhibitor'

Received for publication April 14, 1987 and in revised form May 12, 1987

VINCENT GIRARD2 AND GORDON MACLACHLAN*Department ofBiology, McGill University, Montreal, Quebec, Canada H3A JBI

ABSTRACr

#-Glucan synthase activity in plant membranes can be markedlyaltered by a multiplicity of apparently unrelated factors. In pea epicotylmembranes it is enhanced by low and inhibited by high concentrations ofadded Ca2+, trypsin or soluble pea protease. Ca2` stimulates preexistingsynthase activity, particularly in the presence of polycations (spermidine),but protease treatments activate and, with time, inactivate synthasezymogen. Endogenous pea protease activity is also associated withwashed pea membrane and appears to be responsible for the decayobserved with time in the fl-glucan synthase activity. Endogenous peaprotease activity is inhibited by thiol inhibitors, e.g. iodoacetamide andHg2+, and by a heat-stable peptide, molecular weight approximately10,000, that is found in supernatants of pea extracts. These proteaseinhibitors have the capacity to protect 6-glucan synthase activity fromdenaturation or its zymogen from activation due to endogenous or addedprotease activity. Evidence is described which supports the proposal that1,4-ftglucan synthase is destroyed and possibly converted to 1,3-0-glucansynthase activity by protease action, and that the latter may then begreatly enhanced by Ca2 and polycations.

1,3-fl-Glpcan (callose) of higher plants is often deposited veryrapidly in localized periplasmic spaces after tissues are woundedor stressed, and it disappears quickly if tissues are returned tonormal conditions (3, 8, 11, 19, 25), indicating that it is subjectto turnover by processes that are closely regulated. In manyfungi, in contrast, 1,3-fl-glucan is an integral part of the wallwhich is deposited during extension growth and only degradedduring budding or branching (10). Despite these differences, thegeneration of 1,3-f3-glucan in both plants and fungi appears tobe controlled by endogenous factors that either activate or inhibit1,3-fl-glucan synthase activity. In fungal membranes, 1,3-,B-glu-can synthase may exist as a zymogen which is activated bymoderate proteolysis or inactivated if proteolysis continues (9,24). In soybean microsomes, 1,3-,B-glucan synthase activity iseither stimulated or inhibited by adding suitable concentrationsof trypsin or soybean trypsin inhibitor (14, 16). In soybean (16)and cotton (5, 6) membranes, synthase is also activated by Ca2",in a reaction that can be enhanced by polycations (15) orphospholipids, unsaturated fatty acids, and lysophosphatidylcho-

' Supported by Natural Sciences and Engineering Research Council ofCanada.

2 Prent address: Laboratoire de differentiation fongique associe auCNRS, Universite Lyon 1, 43, Boulevard du 1 I Novembre, 1918, F-69622, Villeurbanne Cedex, France.

line (14). In pea stem membranes, natural regulators may alsoact to control glucan synthase activity since it has been shown(2) to be stimulated by a dialysable heat-stable component ofpeaextracts and inhibited by a soluble heat-labile component thatco-chromatographs with pea protease. The interrelationships ofall of these potential moderators of ,3-glucan synthase need to beclarified, with particular reference to the formation of 1,3-3versus 1,4-, linkages.The present study reexamines the properties of endogenous

pea factors that can modify pea (3-glucan synthase activity.Evidence is presented for potential regulation by Ca2" and poly-cation levels as well as by pea protease and pea protease inhibitor.The results are examined with particular attention to the possi-bility that limited proteolysis perturbs membranes in such a waythat 1,4-f3-glucan synthase is converted to 1,3-,B-glucan synthaseactivity.

MATERIALS AND METHODS

Preparation of Particulate Enzyme. Pea seedlings (Pisum sa-tivum var Alaska) were grown in darkness and the apical 35 mmof third internode was homogenized in buffer (25 mm Pipes-TrispH 7.5, 0.5 M sorbitol) containing 10 mm EDTA, 0.1 mg/mlBSA, and 0.35 mg/ml PMSF.3 In some experiments, 2.5 mMCaCl2 was included in the homogenization medium with orwithout EDTA (or EGTA). Particulate fractions were preparedby homogenizing in a cold mortar, squeezing the homogenatethrough nylon, centrifuging at 2,000g for 10 min, and centrifug-ing the supernatant again at 27,000g for 30 min. The pellet wassuspended in 25 mm Pipes-Tris (pH 7.5) containing 0.5 M sor-bitol. All operations were conducted at 4°C.

i-Glucan Synthase Assays. The standard assay mixture (totalvol 350 ,l) contained 0.25 uCi UDP-['4C]glucose plus 1 mMunlabeled UDP-glucose, 0.1 M Tris buffer (pH 8.0), 5 mM cello-biose, 10 mM MgCl2, 5 mm DTT, and 100 ul freshly isolatedmembrane suspension (0.5-1.0 mg protein). The mixture wasincubated at 27°C for 10 to 60 min and reactions were terminatedby addition of 2 ml ethanol. After addition ofpowdered cellulose(10-20 mg) the mixture was filtered through glass-fiber (What-man GF/C, 2.4 cm). The residue on the filter was washed withwater, methanol/chloroform (2/1 v/v), and ethanol. Radioactiv-ity of the dried filters was measured in aquasol scintillation fluid(NEN) using a Beckman liquid scintillation spectrometer (CPM-100) with 90% counting efficiency.Protease Assay. The enzyme to be assayed (200 ul) was mixed

with 200 ul of 1% casein (w/v) in 0.1 M Tris-HCl buffer (pH 8.0)plus 200 gl water, and incubated at 30°C for 1 h. The reaction

'Abbreviations: PMSF, phenylmethyl sulfonyl fluoride; TLCK, tosyllysine chloromethyl ketone.

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GIRARD AND MACLACHLAN

was stopped by addition of 750 Al of 10% TCA. Control mixtureswere the same with TCA added at zero time. After standing for30 min, the precipitated proteins were removed by centrifuga-tion, 250 gl of supernatant was diluted to 1.0 ml with water, andthe optical density measured at 280 nm. Activity was expressedin O.D. units generated per unit time.

Partial Acid Hydrolysis. Radioactive products in reactionmixtures were collected after addition of ethanol by centrifuga-tion (5000g, 10 min), and subjected to acid hydrolysis by mixingwith 500 Ml of concentrated HCl to which 50 Al of fuming HClwas added at 0°C. Mixtures were incubated at 24°C for 6 h,evaporated in an air stream and the dried samples resuspendedin water and chromatographed on Whatman No. 1 paper usingl-propanol-ethyl acetate-H20 (7:1:2, v/v) as solvent system.Enzymatic Hydrolysis. The air-dried radioactive products were

subjected to enzymatic hydrolysis by cellulase from Trichodermaviride (CSE II, Worthington). Pellets were suspended in 400 Ml0.05 M acetate buffer (pH 5) containing 1 mg/ml cellulase and 3mmol NaN3 to prevent bacterial contamination. Mixtures wereincubated at 40°C for 20 h and reactions were stopped at 100°C.Digests were air-dried, redissolved in a small volume of water,and chromatographed on Whatman No. 1 paper using the samesolvent system as above.Other Procedures. Crude extracts containing soluble pea pro-

tease were prepared by dialysis of the supernatant fraction over-night against 25 mm Tris buffer (pH 7.4) at 4°C. The proteaseinhibitor was obtained by boiling the supematant fraction, cen-trifuging (1000g, 10 min) and concentrating 5-fold by rotaryevaporation. In some tests, this factor was desalted on a Bio-GelP-2 column eluted with H20 in order to eliminate endogenousCa2'. Chromatography of the protease inhibitor was carried outon a Bio-Gel P-6 or P-10 column (1.6 x 26 cm) equilibrated andeluted with water.

Chemicals. UDP-['4C]glucose (1 1.0 GBq/mmol) was obtainedfrom Amersham. Trypsin, chymotrypsin, pepsin, and papain, aswell as leupeptin, BSA soybean trypsin inhibitor, and spermidine,were purchased from Sigma. Bio-Gel type P came from Bio-Rad.All other compounds were purchased from Fisher.

RESULTS

Effects of Calcium, Trypsin, and Spermidine on Pea ,%GlucanSynthase Activity. The incorporation in 1 h of ['4C]glucose from1 mm UDP-glucose into water-soluble products by pea mem-branes was enhanced by the concurrent addition of Ca2" (maxi-mum at 1 mM) or trypsin (optimum 10 ,ug/ml, Fig. 1). Half-maximum stimulation with added Ca2" was observed at about75 Mm, and at lower free Ca2+ concentrations if the levels ofendogenous Ca2" were first reduced by homogenizing mem-branes and incubating in EGTA or EDTA. Both additives wereinhibitory at higher concentrations.

Trypsin treatments were also stimulatory, even at concentra-tions that eventually destroyed synthase activity in early stagesofthe incubation or if membranes were only briefly preincubatedwith the protease (Fig. 2). The trypsin effect was clearly time-dependent. Papain enhanced synthase activity with an optimumconcentration (10 Mg/ml) similar to that of trypsin. Some pro-teases with different specificities, e.g. chymotrypsin or pepsin,were inhibitory at high concentrations but failed to show an

activating effect at their pH optimum.The fact that trypsin effects were time-dependent and persisted

after the enzyme was washed out of the membrane preparation(Fig. 2) implies that the protease activity had brought about anirreversible change in the membranes that led to enhanced glucansynthase activity. The stimulation was not due to the fact thattrypsin is cationic at the pH used in these incubations. Boiledtrypsin had no effect on synthase activity (Table I). Cations likespermidine were actually inhibitory to synthase activity when

300

I~->

z4

3(D-J0

z0C.

Ol

200

lo0o

A

ol I I I I Il5 10 15 20 25

Co2+(mM)

150I~~~~BI100Y0

500 10o0 l I 1l

10 50 100TRYPSIN (pg/ml)

FIG. 1. Effects of increasing levels of Ca2+ and trypsin on ,-glucansynthase activity of pea membranes. Ca2+ (A) and trypsin (B) were addedat the beginning of the reaction. Incubation time was 1 h. ['4C]Glucoseincorporation into insoluble products in controls for both experimentswithout additives was 48,600 cpm.

I-2.5

C.)

31 E

15

10

0 I I I 15 10 15 20

PRE-TREATMENT TIME (min)FIG. 2. Effect of membrane preincubation with typsin (500 M&g/ml)

on #-glucan synthase activity. Preincubation was for various times atroom temperature. Membranes were centrifuged (Ependorf microfuge,15,600g, 15 min, 4C), pellets were resuspended in buffer (protein con-

centration, 0.45 mg/ml) and glucan synthase was assayed (I h incubation)as described in "Materials and Methods."

added without Ca2+ but spermidine enhanced the stimulationdue to Ca2+ (Table I; Fig. 3). Thus, there was synergistic actionbetween effects of Ca2' and trypsin, but not between spermidineand trypsin.Endogenous Proteases and f-Glucan Synthase Activity. Heat-

labile protease activity was readily detectable in pea membranepreparations as well as in supernatant extracts (after centrifuga-tion of homogenates) (Table II). In the presence of casein (1%w/v) at pH 8.0, both the washed membranes and the dialyzedsupernatant generated TCA-soluble products with a high opticaldensity at 280 nm. Specific casein-hydrolysing activities per unitof membrane or supernatant protein were similar (1.2-1.4 O.D.units/mg protein h). Casein hydrolysis by pea membranes or

soluble fractions did not appear to be due to serine-type proteases,

Plant Physiol. Vol. 85, 1987132

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MODULATION OF PEA 3-GLUCAN SYNTHASE ACTIVITY

Table 1. Stimulation ofPea f8-Glucan Synthase Activity by AddedTrypsin, Ca2", and Spermidine

Synthase was assayed as in "Materials and Methods" with or withouttrypsin (10 ug/ml) and other components added along with UDP['4C]-glucose at the start of reaction. Incubation time I h.

(l-Glucan Synthase ActivityAdditives

Minus Trypsin Plus Trypsin

% controlNone (control) 100 182+ Boiled trypsin (25 ,g/ml) 99 169+ Ca2+ (25 uM) 159 280+ Spermidine (50 Mm) 60 160+ Ca2 + spermidine 232 305

= 300e

8

i- 200

w

4

z

(>I)z 100

3IJ0~l

0 50 100 200

CONC. SPERMIDINE (,M)FIG. 3. Effect of added spermidine with or without Ca2" on (l-glucan

synthase activity. Membranes were incubated with various concentrationof spermidine in the absence of Ca2" (0) or in 10 AM Ca2" (0) or 50 AMCa2+ (0)

Table II. Effectors ofProteolytic Activity in Soluble and ParticulateFractions ofPea Epicotyl

Soluble and particulate fractions were isolated and proteolytic activi-ties versus casein were assayed as described in "Materials and Methods."lodoacetamide was preincubated at room temperature with both frac-tions for 15 min before adding casein; all the other reagents were addedconcurrently with substrate.

Proteolytic ActivityReagent Added

Supernatant Particulate

% controlGlutathione (reduced), 5 mm 108 106Ca2+, 1 mm 100 100EDTA, 5 mM 99 94Soybean trypsin inhibitor, 0.25% 98 97PMSF, 0.25 mm 85 86TLCK, I mM 76 75Leupeptin, 0.1 mg/ml 71 68Iodoacetamide, 25 mm 51 42HgCI2,2.5mM 0 13

since neither was inhibited by soybean trypsin inhibitor and bothwere only slightly inhibited by PMSF. However, they were dis-tinctly inhibited by TLCK or leupeptin and especially by iodoa-cetamide and they were abolished by Hg2+. These properties aretypical of papain-like sulfhydryl-dependent proteases. Proteaseactivities with very similar properties have also been detectedfirmly bound to plasmamembranes of pea cotyledons (20). Peaprotease activities were totally unaffected by EDTA or by con-

centrations of Ca2+ (up to 1 mM) which stimulated glucansynthase activity.

Inactivation by endogenous membrane-bound proteases couldhelp to account for the decay of ,l-glucan synthase activity whichis always observed during membrane incubation. Washed mem-branes prepared and preincubated at 27°C without added Ca2+lost over half of their synthase activity within 30 min (Table III).Membranes prepared in the presence of 2.5 mM Ca2+ possessedmuch higher initial glucan synthase activity, especially if theywere also incubated in added 1 mM Ca2+. Nevertheless, abouthalf of this enhanced activity was also lost during a 30 minpreincubation, even in the presence of added Ca2" (Table III).Clearly, Ca2"-activated tl-glucan synthase activity was as suscep-tible to denaturation during incubation as was endogenous syn-thase.When relatively high concentrations of dialyzed pea extract

were added to pea membranes, the endogenous fl-glucan synthaseactivity decayed even more rapidly than in control membranes(Fig. 4). Although earlier studies (2) failed to detect any activatingeffect of pea extracts on synthase activity, in present tests atintermediate concentrations ofadded extract, there was a distinctenhancement of synthase activity. This pattern of response wasreminiscent of the effect of added trypsin, which increased syn-

thase activity at low but decreased at a high concentration (Fig.1B). The absolute magnitude of the synthase enhancement byaddition of dialysed supernatant was slightly less for membranesprepared with than without Ca2". In this test there was a 4-folddifference in synthase activity levels between the two prepara-tions. This implies that the supernatant did not enhance theCa2+-activated synthase activity but, rather, activated a precursorenzyme or unmasked a latent synthase. Pretreatment of thedialysed supernatant with 25 mM iodoacetamide to inhibit thiol-protease activity eliminated both the activating and inactivatingcapacities of dialysed supernatant on synthase activity (Fig. 4).

Heat-Stable Supernatant Factor. When boiled pea supernatantwas added to freshly prepared pea membranes, the (3-glucansynthase activity assayed 30 min later was higher by an amountthat depended on the supernatant concentration (Table IV).Higher yields of activity were observed using membranes incu-bated in the presence of EDTA, as well as with desalted super-

Table III. Decay ofMembrane-bound ,-Glucan Synthase Activity withTime as Affected by Isolation and/or Incubation in I mm CalciumMembranes were isolated as described in "Materials and Methods,"

and preincubated at 27°C for up to 1 h before addition of UDP-['4C]-glucose. Synthase activity was assayed by incorporation of '4C in 10 minfollowing preincubation treatments.

3-Glucan Synthase ActivityPreincubation

Membranes isolated in CaTime No Ca2"

Incubated - Ca2" Incubated + Ca2"min cpm 10-3/lo min and % ofzero time

0 14.8 (100%) 62.9 (100%) 83.6 (100%)5 12.6 (85) 52.8 (84) 71.9 (86)

10 10.5 (71) 54.3 (72) 58.9 (70)20 8.4 (56) 37.5 (60) 46.9 (56)30 6.7 (45) 32.4 (51) 36.8 (44)60 6.3 (43) 25.2 (40) 28.2 (34)

i I I I

133

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GIRARD AND MACLACHLAN

!R

CnI

cn

.c

E

0

e

E0k.u

25 50 75 100 125

DIALYSED SUPERNATANT (pi.)FIG. 4. Effect of pea supernatant on IB-glucan synthase activity. Dif-

ferent amounts of dialysed pea supernatant (0.2 mg protein/ml) wereadded to membranes (1 mg protein) obtained by homogenization of peasegments either in EDTA minus Ca2" (0) or in the presence of addedCa+ (2.5 mm, *). Effects of added supernatant plus iodoacetamide (25mM) are also shown (A). Glucan synthase assays were conducted in theabsence of added Ca2".

natant (excluded volume ofP-2 columns). Accordingly, only partof the stimulation by the factor in boiled extract could beattributed to Ca2" in the supernatant.The boiled supernatant clearly provided a degree of protection

against synthase denaturation in the membranes (Fig. SA), sincethe percentage retention of activity increased the longer mem-branes were preincubated in boiled supernatant before assay.This phenomenon appears to be due to the presence of aninhibitor of the heat-labile component that causes denaturation.It could be a protease inhibitor since membrane protease activityversus casein was totally prevented by the addition of sufficientquantity of boiled supernatant (Fig. 5B).The inhibitor/protectant in boiled supernatant co-eluted on

Bio-Gel P-10 with the only component of the eluate that ab-sorbed at 280 nm (Fig. 6). The elution volume corresponded toa mol wt of approximately 10,000, i.e. close to the dialyzablelimit of the pores in the dialysis tubing used in these studies.This component was a polypeptide, since its biochemical actioncould be destroyed by preincubation with trypsin (Table IV).

Synthases Susceptible to Proteases and Protease Inhibitor.Partial hydrolysis (enzymic or dilute acid) of the products of ,B-glucan synthase under conditions used in present tests producedlabeled glucose and laminaribiose as the major low-mol wtcomponents, with cellobiose barely detectable (Table V). Peamembranes were incubated in the standard system with orwithout added trypsin, Ca2", pea protease or pea protease inhib-itor under conditions where all additives resulted in higher i3-glucan synthase activity at the end of the incubation (Figs. 1-5).Glucose could have derived from either 1,3- or 1,4-linked glucansbut, following all treatments, there was a substantial increase (2-3-fold) in the yields of laminaribiose relative to cellobiose (TableV). Treatments with trypsin and pea protease were particularlyeffective in reducing the yields of cellobiose and treatment withCa2" in increasing the yield of laminaribiose (Table V). It isconcluded that Ca2" and protease treatments resulted in en-hanced synthesis of 1,3-13-glucan in the preparations and thatprotease treatments reduced the synthesis of 1,4-13-glucan.

5F

4w

w

0.cn

w

F

0R

-j

0

_o

100

75

50

25

0

I ~~~~A+ ACTOR

CONTROL

10 20 30 40 50 60PRE-INCUBATION TIME (min)

50 K0 ISO 200

HEAT STABLE FACTOR (,ug protein)

FIG. 5. Effect of the boiled supernatant factor on ,B-glucan synthaseand protease activities. The membrane fractions (A) were preincubatedat 27°C in the absence (0) or presence (0) of added factor (0.2 mgprotein/ml). At different preincubation times, ,B-glucan synthase activitywas assayed (cpm/10 min mg membrane protein). The activities ofcontrols (i.e. not preincubated) were 18,340 and 38,000 cpm/10 min,respectively in the absence and presence of factor. B, Effect of increasinglevels of boiled supematant factor on membrane-bound protease activity(control, 0.8 O.D. units/h. mg protein).

I2

0

U

z

0

z0

10 20 30 40 50 60

0

0

zui0

iE

FRACTIONFIG. 6. Biogel P-10 permeation chromatography of the boiled super-

natant fraction. The distribution of protein (280 nm A, 0) and proteaseinhibitor (0) %'as measured in eluted fractions. Arrows indicate the voidvolume (VO) and the total volume of the mobile phase in the column(V,). The peaks of protein and protease inhibitor coincide at an elutionvolume corresponding to markers of mol wt 10,000.

DISCUSSION

i3-Glucan synthase activity in pea membranes, as in soybean(13, 15, 16) and cotton (5-7), can be increased many-fold by theaddition of Ca2e, particularly in the presence of polycations (Fig.3) or when membranes have been predepleted of endogenous

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MODULATION OF PEA f3-GLUCAN SYNTHASE ACTIVITY

Table IV. Effect ofBoiled Pea Extract on /3-Glucan Synthase ActivityBoiled supernatant from pea homogenate was added at different

concentrations to fresh pea membranes. Effects on the yields of glucansynthase activity were measured using crude extract and desalted extract(the void volume after fractionation on Bio-Gel P-2). Assays are describedin "Materials and Methods." Effects of pretreatment of the boiled extractwith trypsin (1 h, 24C, boiled to inactivate trypsin) were also tested.

f,-Glucan SynthaseBoiled Pea Extract Activity

Crude Desaltedg protein cpm 10-3/h* mg protein

0 23 2020 68 5140 110 7560 128 76

Boiled extract pretreated with trypsin and reboiled100 27 25

Ca2" by chelation (Tables III and V; Figs. 1A and 4). Themechanism of action of Ca2" is not known, but in the presenttests it was not merely stabilizing the synthase or protecting itfrom inactivation (Table III), nor does it appear to act via acalmodulin-mediated step (16) or a Ca2"-activated protein kinase(17). The activation appeared to be confined to reactions thatled to 1,3-fl linkages (Table V).

Callose deposition in vivo is also reduced by chelating agentsand enhanced by Ca2" (17). But in addition, callose is evokednonspecifically by a great number of treatments which chemi-cally or physically wound or stress the plant (3, 8). Such treat-ments may perturb membrane integrity in such a way that cellcompartments become leaky with the result that normal distri-bution of solutes is disturbed. Thus, it has been suggested (14,16) that Ca2" levels in stressed cells increase at the membranesite of 1,3-fl-glucan synthase to sufficient levels to stimulateactivity and result in visible callose deposition.An alternative explanation for wound-induced callose deposi-

tion is the activation of a 1,3-fl-glucan synthase zymogen bydisplaced endogenous protease. As with soybean membranes(16), in pea membranes low conc or brief treatments with addedtrypsin (Figs. 1 and 2) or supernatant containing soluble protease(Table II; Fig. 4) clearly activate ,B-glucan synthase in a time-dependent manner. Boiled trypsin has no such effect (Table I),protease inhibitor prevents the effect (Fig. 4). Polycations onlystimulate the Ca2+-activated system (Fig. 3), whereas trypsin andpea protease enhance activity in the absence of Ca2+ (Figs. 1, 2,and 4). Thus, the protease effect is enzymic and not due to

cationic properties.High levels ofproteases or prolonged exposure lead to eventual

inactivation of synthase. Since soluble proteases in plant cells areoften concentrated in vacuoles, it may be that wounding orhomogenization results in vacuolar protease coming into abnor-mal contact with synthase zymogen in the plasmamembranewith the result that such cells and membrane preparations form1,3-fl-glucan in a transient manner, which they normally do notdo in undisturbed tissues. It is also possible that woundingperturbs plasmamembranes in such a way that membrane-boundprotease and synthase zymogen are able to interact. Variation inprotease location offers yet another potential control point forregulating,B-glucan synthase activity.

Present results demonstrate for the first time that etiolated peastems also contain a natural heat-stable factor that inhibits peaprotease activity and protects 1,3-f3-glucan synthase from inac-tivation (Table IV; Fig. 5). This inhibitor is a polypeptide of molwt 10,000 (Fig. 6), which differs from the soybean proteaseinhibitor in that it has no effect on the serine-type of proteasebut, rather, inhibits plant thiol-protease (e.g. papain), of whichpea protease(s) appears to be an example (Table II). Such pro-teases are widespread in plants and are often partially membrane-bound (20, 23). There is clear evidence, for example, that a thiol-protease in castor bean endosperm is the prime agent responsiblefor the rapid destruction of many enzyme activities when thetissue is homogenized (1). Thiol-protease inhibitors have alsobeen isolated, e.g. from potato (22) and detected in many otherplant sources. Most have a relatively low mol wt, as does the peainhibitor.

This study confirms earlier observations (2) that proteasesquickly destroy the capacity of isolated pea membranes to syn-thesize 1 ,4-,B-linked glucan (Table V). It is now clear that proteasedoes this even while enhancing, albeit transiently, the formationof 1,3-,B-linked glucan. The two effects of protease may beindependent processes, but they also recall a speculation, firstintroduced by Delmer (4) and contemplated since by manyothers (12, 18, 21), that the difficulties which all investigatorshave had in preparing plant membranes containing active 1,4-fl-glucan synthase may be due to its conversion to 1,3-,B-glucansynthase during membrane isolation. If this reaction does indeedtake place and is catalysed by proteolysis, it may be representedas follows:

1,4-j3-Glucan synthase (zymogen) -. limited protease action

1,3-fl-glucan synthase (activated)

prolonged protease action -- inactive synthase

This reaction scheme is consistent with the observations that

Table V. Effect ofAdded Calcium, Proteases, or Protease Inhibitor on Linkages in the Glucan SynthaseReaction Products

Membranes (1 mg protein) were incubated in trypsin, (10 itg/ml), Ca2+ (1 mM), dialysed supernatant(endogenous protease, 0.5 mg protein/ml), or boiled supernatant (endogenous protease inhibitor, mg protein/ml) for a period that led to enhanced synthase activity. Products formed were partially hydrolyzed in acid and,after chromatography, fractions corresponding to peaks of cellobiose, laminaribiose, and glucose were pooledand counted to determine yields.

Fraction Control + Ca + Trypsin + Pea + Protease

Protease Inhibitorcpm incorporated

Total 14020 31480 18220 17980 21970Cellobiose 435 445 280 190 335Laminaribiose 2565 6005 3625 3515 4105Glucose 3605 7830 4960 4805 5795

ratio laminaribiose:cellobiose5.9 13.5 13.0 18.5 12.3

135

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GIRARD AND MACLACHLAN

inhibitors of pea thiol-protease can prevent both the protease-evoked increase and eventual decay of Il-glucan synthase activity(Figs. 4 and 5; Table V). Thus, it is possible that the distributionand levels of natural protease and protease inhibitor act asmoderators of plant (3-glucan synthase activities in vivo. Studiesare underway in this laboratory to employ the appropriate pro-tease inhibitors during and after membrane isolation as potentialprotectants of 1,4-fl-glucan synthase activity and suppressors of1,3-ft glucan synthase activation, in an effort to increase the yieldof the former.

Acknowledgments-We wish to thank Drs. Anne Camirand and Martial Saugyfor valuable assistance during this study.

LITERATURE CITED

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2. CHAO H-Y, GA MACLACHLAN 1978 Soluble factors in Pisum extracts whichmoderate Pisum fi-glucan synthase activity. Plant Physiol 61: 943-948

3. CURRIER HP 1957 Callose substance in plant cells. Am J Bot 44: 478-4884. DELMER DP 1977 The biosynthesis of cellulose and other plant cell wall

polysaccharides. Recent Adv Phytochem I1: 47-775. DELMER DP 1983 Biosynthesis of cellulose. Adv Carbohydr Chem Biochem

41: 105-1536. DELMER DP, G COOPER, D ALEXANDER, J COOPER, T HAYASHI, L NITSCHE,

M THELEN 1985 New approaches to the study of cellulose synthesis. J CellSci Suppl 2: 33-50

7. DELMER DP, V HEINIGER, C KuLow 1977 UDP-glucose: glucan synthetase indeveloping cotton fibers. Plant Physiol 59: 713-718

8. ESCHRICH W 1975 Sealing systems in phloem. In MH Zimmerman, JAMilbum, eds, Encyclopedia of Plant Physiology, Transport in Plants, I.Phloem Transport. Springer-Verlag, Berlin, pp 39-56

9. FEVRE M 1979 Digitonin solubilization and protease stimulation of fB-glucansynthetases of Saprolegnia. Z Pflanzenphysiol 95: 129-140

10. FEVRE M, M ROUGIER 1982 Autoradiographic study of hyphal cell wallsynthesis of Saprolegnia. Arch Microbiol 131: 212-215

1 1. FINCHER GB, BA STONE 1981 Metabolism of noncellulosic polysaccharides. InW Tanner, FA Loewus, ed, Encyclopedia of Plant Physiology, Plant Carbo-hydrates, II. Extracellular carbohydrates. Springer-Verlag, Berlin, pp 68-132

12. JACOB SR, DH NORTHCOTE 1985 In vitro glucan synthesis by membranes ofcelery petioles: the role of the membrane in determining the type of linkageformed. J Cell Sci 2: Suppl, 1-11

13. KAuss H 1985 Callose biosynthesis as a Ca2l regulated process and possiblerelations to the induction of other metabolic changes. J Cell Sci 2: Suppl,98-103

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136 Plant Physiol. Vol. 85, 1987

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