Plant Physiol. (1978) 62, 754-760

Regulation of Glyoxysomal Enzymes during Germination ofCucumber2. ISOLATION AND IMMUNOLOGICAL DETECTION OF ISOCITRATE LYASE AND CATALASE'

Received for publication June 12, 1978 and in revised form July 29, 1978

JAMIE E. LAMB,2 HOWARD RIEZMAN, AND WAYNE M. BECKER3Department of Botany, University of Wisconsin, Madison, Wisconsin 53706

CHRISTOPHER J. LEAVERDepartment of Botany, University of Edinburgh, Edinburgh, Scotland

ABSTRACT

The glyoxysomal enzymes isocitrate lyase and catalase have beenisolated from etiolated cucumber (Cucumis satdvus) cotyledons. The en-zymes co-purified through polyethyleneimine precipitation and (NH4)2SO4precipitation, and were resolved by gel filtration on Sepharose 6B foUlowedby chromatography on diethylaminoethyl-cefulose (isocitrate lyase) orhydroxylapatite (catalase). Purity of the isolated enzymes was assessed bysodium dodecyl sulfate-polyacrylamide electrophoresis, isoelectric focus-ing, and immunoelectrophoresis. Antibodies raised to both enzymes inrabbits and in tumor-bearing mice were shown to be monospecific byimmunoelectrophoresis against total homogenate protein. Isocitrate lyaseand catalase represent about 0.56% and 0.1%, respectively, of total extract-able cotyledonary protein. Both enzymes appear to be present in a singleform. Molecular weights of the native enzymes and its subunits are 225,000and 54,500 for catalase, and 325,000 and 63,500 for isocitrate lyase. ThepH optimum for isocitrate lyase is about 6.75 in morpholinopropanesulfonic acid buffer, but varies significantly with buffer used. The Km forD-isocitrate is 39 micromolar. A double antibody technique (rabbit anti-isocitrate lyase followed by 1251-labeled goat anti-rabbit immunoglobulinG) has been used to visualize isocitrate lyase subunit protein on sodiumdodecyl sulfate-polyacrylamide with high specificity and sensitivity.

Seed germination in fat-storing species requires a functionalglyoxylate cycle to effect net gluconeogenesis from the acetyl-CoAderived by fl-oxidation of storage triglycerides (4). During earlygermination, glyoxylate cycle enzymes such as isocitrate lyase(threo-D-isocitrate glyoxylate lyase, EC 4.1.3.1) undergo a wellcharacterized increase and subsequent decline in activity, withpeak activity corresponding to the period of maximum fat metab-olism (3, 4, 7, 16, 32, 35). Much is already known about thedevelopmental physiology of the glyoxylate cycle enzymes andthe glyoxysomal compartment in which they are localized, butlittle is presently understood about the regulatory mechanismswhich underlie their appearance and subcellular compartmenta-tion in the germinating seed. The same could be said of catalase(H202:H202 oxidoreductase EC 1.11.1.6), another glyoxysomal

'This work was supported by NSF Grant PCM76418051 to W. M. B.,by ARC Grant AG 15/144 to C. J. L., and by a NATO Travel Grant to C.J. L. and W. M. B.

2 Present address: Department of Biological Chemistry, University ofMichigan, Ann Arbor.'To whom reprint requests should be addressed.

enzyme with a characteristic increase and subsequent decline inactivity during germination (35). We are interested both in thelevel(s) at which the activities of glyoxysomal enzymes are regu-lated during cucumber germination (3) and in the relationshipbetween appearance of enzyme activity and organelle biogenesis.Critical to such studies is the availability of purified glyoxysomalenzymes and of monospecific antibodies to them. Few reportshave appeared to date concerning the isolation of glyoxysomalenzymes from plant sources. Specifically, ICL4 has been isolatedfrom bacterial (25), fungal (14), algal (15), and nematode (9, 28)sources, but its purification from angiosperms has thus far beendescribed only for flax by Khan et al. (18) and for cucumber inour own preliminary communication (21) which we now report inmore detail. Similarly, catalase has previously been purified fromonly a few plant sources (13, 31). We report here the purificationand properties ofICL and catalase from cucumber cotyledons andthe preparation of monospecific antisera against both enzymes.An indirect immunological method for the specific detection ofICL on SDS-polyacrylamide gels is also described.

MATERIALS AND METHODS

Sources. Cucumber seeds (Cucumis sativus var. Improved LongGreen) were purchased from L. L. Olds Seed Company, Madison,Wis. Acrylamide and bis-acrylamide were purchased from AldrichChemical Co. and recrystallized according to Loening (22). Bio-Gel HTP and SDS were obtained from Bio-Rad Laboratories.DEAE-cellulose (DE32 advanced fibrous, fines reduced) was aWhatman product, and Polymin-P was obtained from Miles Lab-oratories. Agarose (type III, high EEO) and DL-isocitrate (triso-dium salt, type I; 43.5% D-isocitrate) were purchased from SigmaChemical Co., Freund's adjuvant was obtained from Difco Lab-oratories.

Buffers. Buffer pH values are reported at 25 C. Buffer compo-sitions are as follows: TEMD, 10 mm Tris-HCl (pH 7.0), 1 mmEDTA, 5 mM MgCl2, I mm DTT; TAN, 50 mM Tris-HCl (pH7.4), 0.1% NaN3, 150 mm NaCl; and PBS, 50 mm potassiumphosphate (pH 7.5), 150 mM NaCl; ED/CA, 150 mm ethylenedi-amine, 100 mm citric acid.Enzyme and Protein Assays. The standard ICL assay (10)

4Abbreviations: ED/CA: ethylenediamine-citric acid buffer; HTP: hy-droxylapatite; ICL: isocitrate lyase; IgG: immunoglobulin G; MOPS:morpholinopropane sulfonic acid; PBS: potassium phosphate/NaCibuffer; Polymin-P: polyethyleneimine; SDS-PAGE: sodium dodecyl sul-fate-polyacrylamide gel electrophoresis; TAN: Tris-HCI/NaN3/NaClbuffer; TEMD: Tris-HCI/EDTA/MgCl2/DTT buffer.

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CUCUMBER ISOCITRATE LYASE AND CATALASE

contained 18 mi MOPS (pH 6.8), 6.3 mIM MgCl2, 0.9 mm EDTA,1.75 mm phenylhydrazine hydrochloride, 1.0 mi DTT, 13mM DL-

isocitrate (preadjusted to pH 6.8) and 10 to 100 ,ul of enzyme in a

final volume of 1.0 ml. The formation of glyoxylate phenylhydra-zone was monitored as AA32nm (extinction coefficient: 1.7 x 104M-1 cm-'). One unit of ICL activity is that amount of enzymerequired to degrade I,umol of threo-D.-isocitrate to glyoxylate andsuccinate per min at 30 C. For kinetic assays, the incubationmixture (14) contained 18.4 mm MOPS (pH 6.8), 1.4 mm MgC12,1.62 mm phenylhydrazine hydrochloride, 1.1 mm DTT, variableconcentrations (0.02-0.15 mM) of DL-isocitrate, and 10 to 20,Al ofenzyme in a final volume of 2.5 ml.

Catalase was assayed by the method of Luck (24). The assay

contained 0.135 M K-phosphate (pH 7.2), 0.096% H202, 0.0125%Triton X-100, and 10 to 100yd of enzyme in a fmal volume of 3.0ml. Control reactions without H202 were assayed simultaneously.The first order decomposition of H202 was monitored at 240 nm.

One unit of catalase activity is that amount of enzyme required todegrade 50% of the H202 in 100 sec at 25 C. For both enzymes,specific activity is expressed as activity units per mg of protein.The protein concentrations of column effluents were monitored

photometrically at 280 nm using an LKB Uvicord II UV absorp-tiometer. Approximate protein concentrations were calculatedfrom measured A260 and A230 values using the formula of Kalband Bemlohr (17). The protein assay of Lowry et al. (23) was usedfor more accurate determinations when needed, with BSA as thestandard.

Isolation of ICL and Catalase. Cotyledons from 3-day dark-grown cucumber seedlings were harvested onto dry ice and storedat -80 C until needed. All subsequent operations were carried outat 0 to 5 C. A summary of the extraction and purification proce-

dure is presented in Figure 1. Each step was monitored by SDS-PAGE (20) in addition to the routine enzyme (ICL and catalase)and protein assays.

Cotyledons were homogenized in 2 volumes of TEMD bufferusing a polytron blendor (Brinkmann Instruments) equipped with

CUCUMBER COTYLEDONSI

A. Extroct, filter,and centrifuge

discordpellet

S - I

B. Ppt. with Polymin P

and centrifuge

discordpellet

S - 2

C. Ppt. with (NH4)2 SO4and centrifuge

p- 3 discardsupt.

Dissolve in TEMD

D. Sephorose 6B D'. DEAE - Sephadex

ICL-enriched Catolase-enrichedfractions fractions

IE. DEAE-Cellulose F. Bio-yel HTP

| CATALASE|FIG. 1. Isolation and purification of ICL and catalase from cucumber

cotyledons. After initial blending in a Polytron homogenizer (step A), thetwo enzymes co-purify through Polymin-P treatment (B) and (NH4)2SO4precipitation (C), are partially separated on Sepharose 6B (D), with finalresolution achieved on DEAE-cellulose for ICL (E) and on hydroxylapatitefor catalase (F).

a PT-20 probe. The slurry was filtered through Miracloth and thefiltrate (hereafter called the homogenate) was centrifuged at 15,-OOOg for 30 min. The resulting supernatant (S-1) was filteredthrough Miracloth and was brought to 1.38% (v/v) Polymin-P byslow addition, with stirring, of a 10%1o (v/v) stock, preadjusted topH 7.0. After stirring for 15 min, the solution was centrifuged atl0,OOOg for 20 min and the pellet of Polymin-precipitated proteinwas discarded. To the supernatant (S-2), solid ammonium sulfatewas added slowly, with stirring, to a final concentration of 50%saturation. Stirring was continued for 1 hr, and the enzyme-enriched pellet (P-3) was collected by centrifugation (l0,OOOg, for20 min) and resuspended in TEMD buffer.

This sample was then loaded onto a Sepharose 6B column (80x 2.5 cm) and eluted with TEMD buffer. (In some experiments,partial enzyme separation was achieved by ion exchange onDEAE-Sephadex rather than gel filtration on Sepharose 6B.) Thepeak ICL fractions from the Sepharose 6B column were pooledand loaded onto a DEAE-cellulose column (1.9 x 17 cm) pree-quilibrated with TEMD buffer. The column was eluted with alinear gradient (0-0.2 M) of NaCl in TEMD buffer. The peak ICLfractions were pooled and used as the purified enzyme for bothantibody production and enzyme characterization.The peak catalase fractions from either Sepharose 6B or DEAE-

Sephadex were pooled and concentrated by precipitation withammonium sulfate (50%o saturation). The resuspended pellet wasdialyzed into 0.01 M K-phosphate (pH 7.85), containing I mrmMgCl2. The dialyzed sample was loaded onto a Bio-Gel HTPcolumn (1 x 12 cm) and eluted with a linear gradient (0.01 - 0.2M) of K-phosphate (pH 7.85). The peak catalase fractions werepooled and used as the purified enzyme for both antibody pro-duction and enzyme characterization.Enzyme Homogeneity and Characterization. Homogeneity of

purified ICL was established by several methods, including: (a)electrophoresis on 7.5% and 15% SDS-polyacrylamide gels (20);(b) isoelectric focusing on urea-polyacrylamide gels (pH gradient:3.5-8.5); (c) two-dimensional analysis with isoelectric focusingfollowed by SDS-PAGE (27); and (d) immunoelectrophoresis inagarose (2). For catalase, homogeneity was determined by SDS-PAGE (20) and by immunoelectrophoresis in agarose (2).

Molecular weights of native ICL and catalase were determinedby gel filtration through Sephadex G-200 (1). Subunit mol wtwere assessed by SDS-PAGE.The pH optimum for ICL activity was determined using three

different buffers: 10 mm K-phosphate, 20 mm MOPS, and 150 mMethylene diamine- 100 mm citric acid, each containing 1 mM MgC12and 1 mm EDTA. The pH of the buffers was varied between 5and 9. The pH of the assay solution was measured in the reactionmixture after the reaction had been monitored spectrophotomet-rically for 2 min. For catalase, the pH optimum was assumed tobe 7.2 in K-phosphate (24).The Km of ICL for its substrate, D-isocitrate, was determined

using the ICL assay described by Johanson et al. (14). Km, Vmax,and V/K values were calculated by computer, using a programkindly provided by W. W. Cleland (8).

Preparation of Antisera. Antiserum against ICL was raised inrabbits by two injections of 50 ,ug of purified enzyme in 0.5 ml ofcomplete Freund's adjuvant, with a week between injections.Rabbits were bled from the marginal ear vein 10 and 17 days afterthe second injection. A booster injection of 50 ,ug of ICL inincomplete adjuvant was given 14 days after the second bleeding,and rabbits were bled approximately 7 and 14 days thereafter.Mouse antisera were prepared against both ICL and catalase

by the procedure of Deig (11). Each of five mice was injectedintraperitoneally with 2 jig of purified enzyme in 0.2 ml of 50%(v/v) complete Freund's adjuvant. Four more identical injectionswere administered at 7-day intervals. Four or more days after thelast injection of antigen, each mouse received by intraperitonealinjection 0.5 to 1.0 ml of sarcoma cells (180/TG; see ref. 11)

755Plant Physiol. Vol. 62, 1978

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D. Mathieson in My Grandfather'sWar (Boston Mills, Erin, Ont., 1985),later described the circumstances asMcCrae wrote the poem: "I saw himsitting on the ambulance step, a pad onhis knee. He looked up when I ap-proached, but continued to write.... Idid not interrupt him. He wrote on for5 minutes more. Then as I handed himhis mail, he handed me his pad. Hisface was very tired but calm as hewrote. He looked around from time totime, his eyes straying to [theofficer's] grave. The poem was almostan exact description of the scene infront of us both...."

The horrors of Ypres changedMcCrae forever. Before the battle hewas often described as cheerful andoptimistic, and a lively guest at socialfunctions. Afterwards he became mo-rose, impatient and withdrawn. Heoften went for long rides in the coun-

tryside, with only his horse and dogfor company. Harvey Cushing, inFrance with a medical unit from Har-vard University, knew McCrae well.In his 1941 account, From a Sur-geon s Journal, he wrote: "He gavehis all with the Canadian artilleryduring the prolonged second Battle ofYpres and after, at which time hewrote his imperishable verses. Sincethose frightful days he has never beenhis old gay and companionable self,

but rather has sought solitude."As the pace of the war quick-

ened and Allied casualties mounted,the need for physicians increased.Consequently, and against hiswishes, McCrae was transferred fromhis beloved artillery to the CanadianArmy Medical Corps. He was pro-moted to lieutenant-colonel andgiven charge of No. 3 Canadian Hos-pital, a 1000-bed unit equipped bydonations and manned by volunteersfrom McGill. Osler's only son, Re-vere, served as the hospital's assis-tant quartermaster.

McCrae and his hospital partici-pated in many major Allied cam-

paigns, including the triumph atVimy Ridge and the futile fight in themud of Passchendaele, where RevereOsler was killed. In a 2-week periodduring the bloody Battle of theSomme, his hospital received 4600wounded soldiers.

Bullets and artillery shell frag-ments caused most wounds. Manymen also suffered pulmonary com-plications from exposure to chlorineand mustard gas. Some were burnedby a terrifying new weapon, theflame thrower. Other diseases re-sulted from the squalid living condi-tions trenchfoot, bronchitis,nephritis and psychiatric disorders.

By late 1917, McCrae's own

health was failing badly. He was con-

siderably troubled by asthma, a life-long condition that may have beenexacerbated by the chlorine gas atYpres. Furthermore, he refused tobillet in a house with the officers, butinsisted on sleeping outdoors in a

tent with the enlisted men, even

through the bitterly cold winters.On Jan. 24, 1918, McCrae be-

came the first Canadian honouredwith an appointment as a consultingphysician to the First British Army. Itwas a duty he accepted but could notfulfil because of a severe case ofpneumonia. On Jan. 26 he developedmeningitis and lost consciousness.He died 2 days later.

He was buried in a ceremony at-tended by some of the most impor-tant officers on the Allied side, in-cluding Sir Arthur Currie, theCanadian corps commander. "A sol-dier from top to toe how he wouldhave hated to die in a bed," Cushingwrote. ..... We saw him buried thisafternoon at the cemetery on the hill-side at Wimereux with full militaryhonors a tribute to Canada as wellas to him.... and so we leave him."

Before the burial, some Cana-dian officers tried to find some

"chance winter poppies" to place on

his grave. McCrae would have ap-preciated the effort.E

In Flanders Fields

In Flanders fields the poppies blowBetween the crosses, row on row,That mark our place; and in the skyThe larks, still bravely singing, flyScarce heard amid the guns below.We are the Dead. Short days agoWe lived, felt dawn, saw sunset glow,Loved, and were loved, and now we lieIn Flanders fields.Take up our quarrel with thefoe:To youfrom failing hands we throwThe torch; be yours to hold it high.Ifye breakfaith with us who dieWe shall not sleep, though poppies growIn Flanders fields.John McCrae (1872-1918)

1310 CAN MED ASSOC I l994. 151 (9) For prescribing information see page 1352 -.1310 CAN MED ASSOC J 1994-,151 (9) For prescribing information see page 1352 --->

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CUCUMBER ISOCITRATE LYASE AND CATALASE

of the ICL protein. Purified ICL had a specific activity of 5.8units/mg, representing an over-all purification of 180-fold.

Catalase Purification. Catalase purification is summarized inTable II. The first four steps (A-D) are as for ICL, since the twoenzymes co-purify through the initial stages. After gel filtrationon Sepharose 6B (Fig. 2) only 9.4% of the initial catalase activitywas recovered, since only those fractions (52-60) with high catalaseactivity and relatively low ICL content were pooled. Sepharose6B increased the specific activity of catalase to 354 units/mg,representing an over-all purification of 69-fold through step D.

Final resolution of catalase from contaminating ICL activitywas accomplished by chromatography on hydroxylapatite (stepF), as shown in Figure 4. Approximately 5% of the originalenzyme activity was present in the catalase peak, but less thanone-third of this (1.4% of the original activity) was recovered inthe peak fraction. The fmal specific activity was 5229 units/mg,which represented a 1,025-fold purification.

Progress of Puriflcation. Purification of both ICL and catalasewas monitored by electrophoresis on 15% SDS-polyacrylamide.Shown on the slab gel of Figure 5 is the protein composition offractions at different stages of purification. The progress of ICLpurification is best illustrated by lanes 1 to 5, which indicate theprotein content of fractions after steps A through E, respectively.The high mol wt polypeptides in lane 5 may actually be dimers ofICL and catalase because the mol wt are 125,000 and 103,000,respectively-approximately twice the subunit mol wt of ICL

Table II. Summary of Catalase Purificationa

FRACrION VOLUP.ME ENZYME ENZYME PROTEIN SPECIFIC PURIFI-ACrIVITY RECOVERY CONTENT ACTIVITY CATION

ml units % mg unit/mgHomogenate 177 31214 100 6110 5 1S-1 137 - - 2630 - -S-2 151 18180 58 560 33 6P-3 4.5 16208 52 123 132 266B-pool 37 2941 9.4 8.3 354 69HTP-poold 7.4 1530 4.9 - - -HTP-peak 1.2 434 1.4 0.08 S229 102S

aFor purification scheme, see Fig. 1

bPooled catalase fractions from Sepharose 6B (Fig. 2. fractions S2 - 60)cPooled catalase fractions from Bio-gel HIP (Fig. 4, fractions 22 - 26)dPeak catalase fraction from Bio-gel IITP (Fig. 4, fraction 23)

T

'Ew

0

CO

0 20 30 40 50Froction Number

FIG. 4. Purification ofcatalase on hydroxylapatite. The pooled catalasefractions from a DEAE-Sephadex column (Fig. 1, step D') were pooledand concentrated by precipitation with (NH4)2SO4 (50o saturation). Theresuspended pellet was dialyzed into 0.01 M K-phosphate (pH 7.85)containing I mM MgCl2. The dialyzed sample was loaded onto a Bio-GelHTP column (1 x 12 cm) and eluted with a linear gradient (0.01-0.2 M) ofK-phosphate (pH 7.85), at a flow rate of about 16 ml/hr. The effluent wasmonitored for protein at A280. Fractions of 1.5 ml were collected andassayed for ICL and catalase. Catalase eluted at about 0.032 M and ICLat about 0.08 M K-phosphate.

90

67

57.5

48.5

400 0

*. x

300

1612

1 2 3 4 5 6 7

FIG. 5. Progress of purification and homogeneity of ICL and catalaseon SDS-polyacrylamide slab gels. Fractions from each stage of the puri-fication procedure (Fig. 1) were subjected to electrophoresis on 15% SDS-polyacrylamide slab gels, as described in the text. The direction of migra-tion is downward toward the anode. Lanes contain: (l) S-1, (2) S-2, (3) P-3; pooled ICL fractions from (4) Sepharose 6B and (5) DEAE-cellulose;pooled catalase fractions from (6) DEAE-Sephadex and (7) hydroxylapa-tite. Mol wt are based on marker proteins run in flanking lanes (notshown).

(63,500) and catalase (54,500). The progress of catalase purifica-tion is illustrated by lanes 1, 2, 3, 6, and 7 of Figure 5 whichcorrespond, respectively, to steps A, B, C, D', and F.

Criteria of Homogeneity. The homogeneity of isolated ICL andcatalase was established by SDS-PAGE (Fig. 5). The single sub-unit band in lane 5 attests to the homogeneity of ICL after DEAE-cellulose, while lane 7 illustrates the purity of catalase afterseparation from ICL or HTP. Both enzymes were determined bydensitometric scanning of the gels to be at least 90% pure.The results of isoelectric focusing in denaturing urea gels are

presented in Figure 6. The left gel illustrates the number ofseparable proteins present in a glyoxysomal preparation isolatedby density gradient centrifugation of a 3-day cotyledonary ho-mogenate, and the right gel shows pure ICL. A major protein isvisible at approximately pH 5.1, with a minor, slightly more basicprotein immediately below it. When purified ICL was subjectedto two-dimensional analysis (isoelectric focusing followed by SDS-PAGE), a single protein was detected (gel not shown).

Immunoelectrophoresis was used to determine both the homo-geneity ofthe purified enzymes and the specificity ofthe antiserum

Plant Physiol. Vol. 62, 1978 757

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LAMB ET AL. Plant Physiol. Vol. 62, 1978

K L -

_:

Ct =

v _.w ... _. .. ... " ... ..... ..Pl _wv _: ... _ - ..... _-f _

5 _...._ _...* _ _. .. . . --v ,w4 ... _ .-,,l - ...et^,_ _: .. ^-. .w_ . .f_ _... _ .. .. ._:::... i .| .... _ .|£ :.... .... . ...-ffi *sa:sS

i

tF

*.:

44

FIG. 6. Isoelectric focusing of ICL. Urea isoelectric focusing gels were

loaded with 100 ug of total glyoxysomal protein (left gel) or about 2 pg ofpurified ICL (right gel) and a constant voltage of 200 v was then main-tained for 16 hr. The resulting pH gradient was from 3.5 (top; anode) to8.5 (bottom; cathode). Gels were fixed in two washes of l1o0 trichloroaceticacid and stained with 0.1% Coomassie brilliant blue.

raised against each. Antibody specificity is illustrated in Figure 7,which shows a single precipitin line between total homogenateprotein (both wells) and either anti-ICL (left trough) or anticata-lase (right trough) antiserum. The center trough contained equalvolumes of both antisera, giving rise to two distinct, nonoverlap-ping precipitin lines against total homogenate protein. Lack ofdetectable reaction against other cotyledonary proteins argues

strongly for the monospecificity of the antiserum and in turn forthe homogeneity of the antigens (the purified enzymes). Antigenhomogeneity was confirmed by the presence of a single precipitinline between purified ICL and antiserum raised against totalglyoxysomal protein (plate not shown).

Further evidence for the monospecificity of the rabbit anti-ICL

FIG. 7. Immunoelectrophoretic demonstration of antibody specificity.Total homogenate protein from 3-day cucumber cotyledons was placed inboth sample wells of an agarose plate and subjected to electrophoresis.The troughs were then filled with mouse anti-ICL (left) or anti-catalase(fight) antiserum, or a mixture of the two (center). Immunodiffusion was

allowed to proceed for 24 hr at 25 C before the plate was photographed.

antiserum is present in Figure 8, in which a polyacrylamide gel oftotal cotyledonary homogenate (on left) has been "stained" forICL by the double antibody technique of Burridge (6). The singleband in the autoradiograph (on right) attests to both the sensitivityof the technique and the specificity of the rabbit anti-ICL antise-rum.

Enzyme Properties. Properties of ICL and catalase are sum-

marized in Table III. ICL and catalase have native mol wt of325,000 and 225,000, respectively, as determined by gel filtrationthrough Sephadex G-200. On SDS-PAGE, ICL yielded a singleband with a subunit mol wt of 63,500 and catalase ran as a singleband at 54,500. Catalase appears to consist of four subunits eachwith a mol wt of 54,500. ICL appears from these data to consist offive similar subunits, though this seems unlikely in light of thetetrameric nature of the enzyme from other sources (14, 15, 18,26).The dependence of ICL activity on pH is depicted in Figure 9

for both MOPS and ED/CA buffers. Noteworthy is the significanteffect of buffer system upon the apparent pH optimum of theenzyme, as well as the 10-fold lower level of activity in ED/CAversus MOPS. The pH optimum ofICL activity was 7.7 in ED/CA,6.75 in MOPS, and 7.0 in K-phosphate (data not shown). Thesubstrate concentration was 13 mM DL-isocitrate in each assay; thisis more than 300 times the Km and should therefore be saturating.A double reciprocal plot for ICL is illustrated in Figure 10. The

Km was determined by computer analysis to be 39 ± 3 UM D-

isocitrate (at pH 6.8 in MOPS) and the Vma,s was 1.200 ± 0.042pmol/min.

758

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Plant Physiol. Vol. 62, 1978 CUCUMBER ISOCITRATE LYASE AND CATALASE 759

310 7 8 9 1

t4

C

w

FIG2

0

3 4 5 6 7 8 9 10pH

FIG. 9. pH optimum of ICL. ICL activity was measured as a functionof pH in 150 mM ED/CA (0) and in 20 mM MOPS (9), both containingI mM MgC12, I mm EDTA, and 13 mM DL-isocitrate. The pH value ofeach assay solution was determined after the reaction had been monitoredspectrophotometrically for 2 min. For the ED/CA data, the enzyme unitsmust be divided by 10 despite the presence of equal amounts of enzymein each assay solution.

FIG. 8. Detection of ICL on SDS-polyacrylamide by double antibodytechnique. Total homogenate protein (75 ,ug) from 3-day cucumber coty-ledons was subjected to slab gel electrophoresis on 15% SDS-acrylamideas described in Figure 5. The gel strip on the left was stained with 0.1%Coomassie brilliant blue. On the right is an autoradiograph obtained byfLxing and overlaying an identical but unstained gel strip as described inthe text. The dried gel was exposed to x-ray film for 10 hr.

Table III. Properties of Isocitrate Lyase and Catalase

Isocitrate Lyase Catalase

Molecular weighta 325,000 225,000Subunit mol. wt. 63,500 S4,500pH optimum in MOPS 6.75

in Pot. Phosphate 7.0in ED/CA 7.7

K 0.039 oMm D-isocitrate

aBy gel filtration on Sephadex G-200

By electrophoresis on SDS-polyacrylamide

DISCUSSION

ICL and catalase from etiolated cucumber cotyledons are ap-

parently quite similar in chemical and physical properties, sincethey co-purify through the initial steps of the isolation procedure(Polymin precipitation, ammonium sulfate precipitation; partialresolution on Sepharose 6B). It was in fact the persistent presence

of catalase in early ICL preparations that caused us to expand our

initial ICL purification efforts to include catalase also. Resolutionof the two enzymes was eventually achieved by ion exchange onDEAE-cellulose (ICL) and fractionation on hydroxylapatite (cat-alase).The final recovery of ICL was about 17% of initial homogenate

activity. This compares favorably with the 14% recovery reportedfor flax seedlings (18), the only other angiosperm source fromwhich the enzyme has to date been isolated. Based on a finalspecific activity of 5.8 units/mg and an over-all purification of180-fold, cucumber ICL represents about 0.56% of extractable

5.0

4.0

* 3.0

1-12

:1,2.0

-40 -20 0 20 40 60 80 100 120

I/S (mM)1FIG. 10. Double reciprocal plot for ICL activity. ICL activity was

measured in MOPS (pH 6.8) at substrate concentrations ranging from0.009 to 0.065 mM D-isocitrate. The graph was constructed by least squaresanalysis of the reciprocals of enzyme activity (jmol/min) and substrateconcentration (mM D-isocitrate), using a computer program.

cotyledonary protein. This seems high, but is similar to the valueof 0.88% that can be calculated from the data for flax seedlingICL (18). For catalase, the over-all recovery was 5%, about halfthat reported for lentil leaf (31). The cucumber enzyme waspurified 1,025-fold to a specific activity of 5229 units/mg, andrepresents about 0.1% of extractable cotyledonary protein.

Purity of the isolated enzymes was established by SDS-PAGEfor both catalase and ICL, and isoelectric focusing, both with andwithout subsequent SDS-PAGE, for ICL. Mutual though tracecontamination of both enzymes could be detected electrophoreti-cally, but only by deliberately overloading the SDS gels. Densi-tometric scanning indicated both enzymes to be at least 90% pure.A single major protein was seen at pH 5.1 when purified ICL wassubjected to isoelectric focusing, with a minor component at aslightly more basic position, possibly attributable to trace contam-ination by catalase. Further evidence of enzyme purity was pro-vided by immunoelectrophoresis, which yielded a single precipitin www.plantphysiol.orgon July 1, 2018 - Published by Downloaded from

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760 LAMB ET AL. Plant Physiol. Vol. 62, 1978

line between either ICL or catalase and antiserum raised againsttotal homogenate protein.Cucumber catalase has a mol wt of 225,000 on Sephadex G-200

and runs on SDS-polyacrylamide with a subunit mol wt of 54,500.Very similar values have been reported for lentil leaf catalase (31);catalase from both sources appears therefore to consist of foursubunits of equal size. The subunit mol wt of 63,500 for ICL isalso in good agreement with literature values (18, 19). The mol wtof 325,000 determined for the native cucumber enzyme is substan-tially higher than most literature values, including 170,000 forChlorella (15), 222,000 for Pseudomonas (26), 265,000 for Neuro-spora (14), and 264,000 for flax (18), although a mol wt of 480,000has been reported for ICL from the vinegar eelworm, Turbatrixaceti (28). The value of 325,000 appears reliable, since it wasdetermined simultaneously with that for catalase, using an appro-priate range of mol wt markers. Unless the enzyme displaysanomalous hydrodynamic properties on Sephadex G-200, cucum-ber ICL does not seem to fit the literature pattern of a tetramericenzyme (14, 15, 18, 26).The pH optimum for cucumber ICL varies significantly with

the choice of buffer (7.7 in ED/CA, 7.0 in K-phosphate, 6.75 inMOPS). Neurospora crassa ICL was also initially reported byJohanson et al. (14) to have a pH optimum of 6.8 in MOPS. Thishas been corrected to 7.4 in a recent report from the samelaboratory (29), with the suggestion that the earlier estimate wasin error due to nonsaturating substrate concentrations. This almostcertainly does not apply to the pH optima determined here, sincethe concentration of ICL used in our assays (13 mm) was at least300 times higher than the Km (0.039 mm). The pH optimum of 7.0in K-phosphate is lower than the 7.5 reported for flax seedlingICL in the same buffer (18). Cucumber ICL appears to differ fromthe Neurospora and the flax seedling enzymes with respect to bothsize and pH optimum.No evidence was found for charge heterogeneity of cucumber

ICL, as has been reported for ICL from flax seedlings (18) andfrom Candida tropicalis (34). Cucumber ICL consistently elutesfrom DEAE-cellulose as a single symmetrical peak, at a muchlower NaCl concentration (0.05 M) than that reported (18) for flaxseedling ICL-I (0.142 M) or ICL-II (0.17 M). Catalase also appearsto be present in a single form in etiolated cucumber cotyledons,since the enzyme elutes from hydroxylapatite as a single sym-metrical peak and runs as a single polypeptide in SDS-polyacryl-amide. In this respect, cucumber catalase resembles that of lentilleaves (31) but differs from the catalases of mustard seedlings (12),maize endosperm (30), and wheat seedlings (33), inasmuch as eachof these sources reportedly contains multiple forms of the enzyme.

Antisera were raised against ICL and catalase in both rabbitsand mice and were shown to be monospecific by immunoelectro-phoresis against total homogenate protein. The use of mice as asource of immune ascitic fluid (11) makes it possible to raiseantibody using a little as 10 ,g of antigen per mouse. With care,multiple withdrawals of ascitic fluid are possible, and 20 to 25 mlof cell-free immune fluid can be obtained per animal in this way.The double antibody technique of Burridge (6) lends itself well

to the autoradiographic visualization of ICL subunit protein inSDS-polyacrylamide gels. Detection of the denatured enzymedoes not appear to be hampered by use ofantiserum raised againstnative ICL. Detection is quite sensitive (less than 100 ng of ICLon the gel of Fig. 8) and highly specific, showing virtually none ofthe nonspecific interactions frequently encountered in immuno-precipitation. Because the second (goat) antiserum is directedagainst the immunoglobulin component of the first (rabbit) anti-serum, it should be readily possible to extend the technique toother glyoxysomal enzymes for which antisera are available or canbe prepared.

This work on the isolation and immunological detection of ICLand catalase complements recent reports of similar success withother glyoxysomal enzymes, including malate synthase (5, 19) andmalate dehydrogenase (36). Availability of purified enzymes and

monospecific antibodies ought to facilitate greatly studies onenzyme regulation and glyoxysome biogenesis during germina-tion.

Acknowledgments--The competent technical assistance of A. Vuchetich and E. Zeldin isgratefully acknowledged.

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