Plant Physiol. (1981) 68, 1339-13440032-0889/81/68/1339/06/$00.50/0

Proteolytic and Trypsin Inhibitor Activity in Germinating JojobaSeeds (Simmondsia chinensis)'

Received for publication April 30, 1981 and in revised form July 17, 1981

DEBORAH SAMAC AND RICHARD STOREYDepartment of Biology, The Colorado College, Colorado Springs, Colorado 80903

ABSTRACT

Changes in proteolytic activity (aminopeptidase, carboxypeptidase, en-dopeptidase) were followed during germination (imbibition through seedlingdevelopment) in extracts from cotyledons of jojoba seeds (Simmondsiachinensis). After imbibition, the cotyledons contained high levels of sulfhy-dryl aminopeptidase activity (APA) but low levels of serine carboxypepti-dase activity (CPA). CPA increased with germination through the apparentloss of a CPA inhibitor substance in the seed. Curves showing changes inendopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 duringgermination were distinctly different. EPA at pH 4, 5, 6, and 7 showedcharacteristics of sulfhydryl enzymes while activity at pH 8 was probablydue to a serine type enzyme. EPA at pH 6 was inhibited early in germinationby one or more substances in the seed. Activities at pH 5 and later at pH6 were the highest of all EPA throughout germination and increases inthese activities were associated with a rapid loss of protein from thecotyledons of the developing seedling.

Jojoba cotyledonary extracts were found to inhibit the enzymic activityof trypsin, chymotrypsin, and pepsin but not the protease from Aspergillussaotoi The heat-labile trypsin inhibitor substance(s) was found in commer-cially processed jojoba seed meal and the albumin fraction of seed proteins.Trypsin inhibitor activity decreased with germination.

The female jojoba shrub (Simmondsia chinensis [Link] Schnei-der) produces numerous seeds which contain about half of theirfresh weight as a unique liquid wax (13). Primarily because of theeconomic value of this liquid wax, the native Sonoran Desertplant is the subject of intense study to improve productivity incultivation (13, 34). The uniqueness of the liquid wax (13, 19)makes the plant an interesting biological system for study andseveral workers have reported on the biochemistry and physiologyof lipid metabolism in the developing and germinating seed (19,21 and references therein). As part of a comprehensive investiga-tion of nitrogen metabolism in jojoba we have reported prelimi-nary results concerning the nature of the possibly unique proteinsin the seed (28).Very little is known about protein catabolism in oilseeds, espe-

cially in those from desert ecosystems. Thus, we have monitoredthe temporal changes in jojoba cotyledonary protein contentduring germination and studied the proteolytic systems responsi-ble for reserve protein degradation during this ontogenetical proc-ess. This paper presents the results of our efforts to identify and

'Supported by grants from Research Corporation and The ColoradoCollege Faculty Research Board.

characterize individual EPA,2 APA, and CPA, and protease inhib-itor activities in the germinating jojoba seed. Preliminary resultsof some of this work have been reported (28).

MATERIALS AND METHODS

Plant Material. Jojoba seeds (Janca's Jojoba Oil and Seed Co.,Mesa, AZ) of uniform size were soaked ovemight in running tapwater, surface sterilized in 5% (v/v) NaOCl, then rinsed in distilledH20 and dusted with Phaltan fungicide. Treated seeds were sownin sterile, moist vermiculite (day 1) and allowed to germinate inthe dark, at 30°C. No fungal or bacterial contamination wasobserved in the germinating seeds. For convenience, germinationis defined as the time from imbibition of the dry seed throughradicle emergence to seedling development for 30 days.

Preparation ofCrude Enzyme Extracts for Assays of ProteolyticActivity (Endopeptidase, Aminopeptidase and Carboxypeptidase)in Germinating Jojoba Cotyledons. Uniform seedlings were har-vested at 3-day intervals; the cotyledons were removed andchopped with a razor blade. The minced cotyledons were homog-enized with a Polytron Homogenizer (speed setting 8) for 1 min in8.0 ml/g fresh weight 25 mm citrate-phosphate buffer containing5.0 mm ME (pH 7.2). The homogenate was filtered throughMiracloth and centrifuged at 10,000g for 10 min. The resultingsupernatant was used directly for endopeptidase and aminopep-tidase assays. For carboxypeptidase assays, the supernatant wasdialyzed overnight against the above buffer. All preceding stepswere carried out at 0 to 5°C. Duplicate assays for each enzymewere conducted on two separate extractions. Results were repro-ducible.Endopeptidase Assay. EPA was assayed with Azocoll (Calbi-

ochem) as the substrate. Reaction mixtures contained 2.0 mlextract and 5.0 mg Azocoll in 2.0 ml 0.1 M citrate-phosphate buffercontaining 5.0 mM ME. Assays were conducted at pH 4, 5, 6, 7,and 8 as determined at the beginning and end of each assay. Thereaction was incubated at 45°C for 2 h with vigorous shaking(about 250 rpm). The reaction was terminated by removing theundigested substrate through centrifugation (7) followed by theaddition of an equal volume of 4.0%o (w/v) Na2CO3 in 2.0 NNaOH to dissolve any remaining reserve proteins and clarify thesupernatant (7). The absorbance of the clarified supernatant wasread at 520 nm. Appropriate controls, including those for turbidityand of substrate alone, were employed. Enzyme activity was linearwith time and volume of extract in the reaction mixture.

Azocoll digestion with subsequent release of peptides bound toa red dye has been widely used in studies to characterize endo-peptidases of both plant and animal cells (5, 7, 18, 25, 33 and

2Abbreviations: EPA, endopeptidase activity; APA, aminopeptidaseactivity; CPA, carboxypeptidase activity; ME, 2-mercaptoethanol; pCMB,p-chloromercurobenzoate; NEM, N-ethylmaleimide; PMSF, phenyl-methylsulfonylfluoride.

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SAMAC AND STOREY

references therein). The primary advantages of this assay systemare its speed and convenience (18). Other workers have also foundAzocoll assays to be sensitive to low levels of activity, linear withtime and enzyme concentration, and reproducible when conductedwith vigorous shaking (18, 25, 33). Furthermore, comparativecharacterization studies of proteinases using traditional physiolog-ical and artificial substrates and Azocoll have yielded similarresults (5, 27).

Aminopeptidase Assay. Leucine APA was assayed by a methodmodified from Chrispeels and Boulter (7). The reaction mixtureconsisted of 0.2 ml jojoba seed extract with 2.3 ml of 2.2 mM L-leucine-p-nitroanilide (final concentration 2.0 mM) in 50 mm cit-rate-phosphate buffer (pH 7.0). The mixture was incubated for 30min at 40°C in a shaking water bath. The reaction was stopped bythe addition of 1.0 ml 1.5 N HC104. Any precipitate was removedby centrifugation and the absorbance read at 410 nm. Appropriatecontrols for turbidity and nonenzymic release of color were em-ployed. APA was directly proportional to time and volume ofextract in the assay mixture.

Carboxypeptidase Assay. The assay for CPA was based on thatof Mikola and Kolehmainen (17). The reaction mixture consistedof 0.1 ml dialyzed jojoba seed extract and 1.9 ml 2.0 mM N-carbobenzoxy-L-phenylalanyl-L-alanine in 50 mm citrate-phos-phate buffer (pH 5), containing 0.5 mm EDTA. After a 30-minincubation in a shaking water bath at 30°C, the reaction wasstopped by the addition of 2.0 ml TNBS reagent (3 volumes of 5%[w/vJ sodium tetraborate to 1 volume 0.2% [w/vJ 2,4,6-trinitro-benzene sulfonic acid). After 1 h shaking at 30°C, the TNBSreaction was stopped by the addition of 1.0 ml 1.5 N acetic acidand any precipitate removed by centrifugation. The absorbanceof the supematant was read at 340 nm. Absorbance of the controlswas subtracted from the experimental values to determine activitywhich was linear with time and enzyme extract in the reactionmixture.Chemical Inhibitor Studies to Characterize Peptidases. The

effects of some common chemical enzyme inhibitors on EPA,APA, and CPA were conducted to characterize these enzymes.Stock solutions of inhibitors were prepared as follows. The sulfhy-dryl inhibitors used were pCMB, a reversible mercaptide-formingreagent, and NEM, an irreversible alkylating reagent (4). ThepCMB was dissolved in 0.3 N NaOH, brought to the desired pH,and diluted to a concentration of 2.0 mm with 0.1 M citrate-phosphate buffer of the desired pH (pCMB was not soluble oractive below pH 6). NEM was dissolved directly in the samebuffer. To ascertain that inhibition by pCMB was due to thepresence of the inhibitor, cysteine (1.0 mM) was used to reverseinhibition. ME (5.0 mM) was also used to determine whetheroptimum enzymic activity was dependent on the presence ofsulfhydryl groups. The serine protease inhibitor, PMSF, was dis-solved in isopropyl alcohol at a concentration of 50 mm anddiluted with the above buffer to a concentration of 2.0 mm (7).Isopropyl alcohol (2%, v/v) was added to controls wheneverappropriate. Metal ion chelators used were EDTA and 1, 10-phen-anthroline. The latter was dissolved in absolute ethanol at aconcentration of 50 mM and diluted to 2.0 mm prior to use with0.1 M citrate-phosphate buffer of the desired pH. EDTA wasdissolved directly in the same buffer. Magnesium chloride, a heavymetal inhibitor ofplant proteases, was dissolved in the same bufferprior to use at a concentration of 2.0 mm. Final concentrations ofall inhibitors in the assay mixtures were 1.0 mm with the exceptionofNEM at 3.0 mm and ME at 2.5 mm.

Protease inhibitor assays were carried out on extracts from daysshowing peak activity during germination. The extract was pre-pared as above except that ME was deleted from the homogenizingbuffer. Endogenous metabolites were removed from the post-10,OOOg supernatant by gel filtration through Sephadex G-25according to the method of Storey and Beevers (30). Prior to

assays, inhibitors and gel-filtered extracts were preincubated to-gether at 5°C for 12 h, then warmed to reaction temperature. TheAPA and CPA assays were conducted as described earlier. EPAwas assayed as above except that the terminated reaction mixturesupernatant was clarified by the addition of an equal volume ofethanol:ether (3:1, v/v).

Assays for Endogenous Inhibitors of Jojoba Seed ProteaseActivity. The possible presence of endogenous inhibitors of EPA,APA, and CPA in the germinating jojoba cotyledons was assayedby standard mixing experiments (3, 29). Equal volumes of gel-filtered extract from 3- and 37-day postimbibition cotyledons weremixed and then preincubated at room temperature for 15 min.ME (2.5 mM) was added to the assays ofenzymes shown to requiresulfhydryl groups for optimal activity. Substrate was added andthe assays were conducted as described above. An expected valueof additive activity was calculated from control assays of the 3-and 37-day extracts alone. A measured activity of the mixedextracts that was lower than the expected additive activity wastaken to indicate the presence of an endogenous inhibitor (3, 29).

Protein. Samples of the crude homogenate were prepared andanalyzed for protein as described previously (30).

Assays for the Influence of Endogenous Jojoba Seed Inhibitorson Proteinases from Other Organisms. Jojoba seed extracts wereassayed for the presence of endogenous inhibitors of bovinetrypsin (Worthington Biochemicals, code TRL), bovine alpha-chymotrypsin (Sigma, bovine pancrease type II), pepsin (Sigma,porcine mucosa) and fungal protease (Aspergillus saotoi, Sigma,type XIII).

Trypsin inhibitor activity was assayed by mixing 2.0 ml com-mercial trypsin (2.5 iLg/ml dissolved in 0.0001 N HCI, 180 A520/h.mg) with aliquots ofjojoba seed extracts. The mixture of trypsinplus seed extract was brought to 4.0 ml with 0.1 M citrate-phos-phate buffer (pH 8.3), then incubated at room temperature for 15min. The reaction was initiated by the addition of Azocoll to theincubation mixture and the assay for trypsin activity was con-ducted as described above for EPA in jojoba seeds (see also thebrochure on Azocoll published by Calbiochem). The followinggel-filtered seed extracts were assayed for trypsin inhibitor activity:(a) extracts from 6- and 37-day postimbibition cotyledons, pre-pared as described above; (b) commercially processed jojoba meal(Janca's Jojoba Oil and Seed Co., Mesa, AZ) prepared in the samemanner as the cotyledon extract; (c) jojoba seed liquid wax (13,19); and (d) jojoba seed albumin and globulin fractions (28).Commercial soybean trypsin inhibitor (Worthington, code SI) wasused as a comparison in proteinase inhibition studies. Assaymixtures containing 1% (w/v) sucrose or 1% (w/v) NaCl insteadof extract were used as additional controls.Chymotrypsin inhibitor activity was assayed in the same man-

ner as above using 20.0 jg/ml chymotrypsin dissolved in 0.001 NHCI andjojoba seed extract from 9-day postimbibition cotyledons.The mixture was brought to 4.0 ml with 0.1 M citrate-phosphatebuffer (pH 8).

Pepsin inhibitor activity was assayed as above, using 20.0 ,ug/ml pepsin dissolved in 0.01 N HCI and jojoba enzyme extract from9-day postimbibition cotyledons. The mixture was brought to atotal volume of 4.0 ml with 5% (v/v) trichloroacetic acid for afinal pH of 1.8.

Inhibitor activity against the protease from Aspergillus saotoiwas measured in the same manner using 1.0 mg/ml proteasedissolved in 0.1 M citrate-phosphate buffer (pH 3) and brought to4.0 ml with the same buffer. The jojoba seed extract was preparedas described above from seed soaked 18 h in distilled H20 at 5°C.

RESULTS

Protease Assays. EPA at pH 4, 5, 6, 7, and 8 were monitoredduring germination. The distinctly different curves showing EPAassayed at each pH value as a function of days postimbibition are

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Plant Physiol. Vol. 68, 1981

shown in Figure 1. EPA at pH 4 was relatively low at the onset ofgermination, then increased 2.5-fold to a peak on day 18 anddeclined slightly thereafter (Fig. 1). EPA at pH 5 was also rela-tively low initially, but activity doubled by day 9 and again afterday 24 (Fig. 1). Peak activity at pH 5 occurred on day 27 and thenfell sharply (Fig. 1). EPA at pH 6 was very low at the onset ofgermination but activity increased gradually through day 21 fol-lowed by a rapid 3-fold increase to a maximum at day 27 whichfell slightly thereafter (Fig. 1). EPA at pH 7 was relatively low atthe beginning of germination and did not change greatly duringgermination (Fig. 1). Small peaks of neutral EPA were detectedon day 9, 18, and 27. EPA at pH 8 decreased from a level equalto EPA at pH 4 immediately following imbibition to very lowlevels which fluctuated slightly through the later stages of germi-nation. (Fig. 1)EPA at pH 5 and later at pH 6 was higher throughout germi-

nation than EPA at the other pH levels tested (Fig. 1). In general,-SH EPA was severalfold higher than neutral or alkaline EPAduring germination (Fig. 1).APA (Fig. 2) was very high at the onset of germination and,

after an initial decline, increased to a maximum at day 15. APAthen dropped precipitously and remained relatively low in laterstages of germination (Fig. 2). In contrast, CPA was relatively lowfollowing imbibition but increased rapidly to a peak at day 12 ofgermination. Activity then decreased steadily to day 21 and re-mained constant in later stages of germination (Fig. 2).The optimum pH values for APA and CPA were 7.0 and 5.0,

respectively (data not shown; see also 7, 11) and activity wasfollowed at these pH values throughout germination. Proteolyticactivity was not assayed in nonimbibed seeds due to the extremehardness of the testa and cotyledons which prevented the custom-

C)0

wCO)

I

'Urco

C3p0U'U0.00z'U

FIG. 1. Change in ]

conducted on extracts"Materials and Metho7 () and pH 8 (A). (days after soaking dryeach pH. The pH was

linear and reproducibli

250

200

150

1006 12 18 24 30

150

0w

CD,

0r

cxlU

50 0

50

DAYS POST-IMBIBITION

FIG. 2. Changes in APA (0) and CPA (U) in germinating jojobacotyledons. Assays were done on crude enzyme extracts of cotyledons asdescribed. Days postimbibition is days after soaking dry seeds (day 0).Data shown are the result of triplicate assays for each substrate on daystested. A/h seed = activity per cotyledon pair.

a'uco2.I-

zax

C:oLLC,

6 1 2 1 8 24 30 50

DAYS POST-IMBIBITIONFIG. 3. Changes in protein and fresh weight in germinating jojoba

cotyledons. Results reported as amount per cotyledon pair (testa and axisremoved). Days postimbibition is days after dry seed was soaked indistilled H20 (day 0).

ary preparation ofenzyme extract.Changes in Protein and Fresh Weight in Germination. Follow-

ing imbibition, the radicle emerged and the hypocotyl elongatedto an average length of 1.5 cm at day 6, 7.5 cm at day 12, 12 cmat day 18, 14 cm at day 24 and 17 cm at day 30. On day 12 the

H 8 pH 7 epicotyl emerged and increased to a height of 16 cm by day 30.During germination, protein decreased and fresh weight in-

creased almost 3-fold (Fig. 3). In the first 12 days of germination,24% of the original protein was lost from the cotyledons. Between

6 1 2 1 8 24 30 day 15 and 30 another 24% of the original protein was lost (14%of that between day 27 and 30. Between day 30 and 50 only 8%

DAYS POST-IMBIBITION protein was mobilized from the cotyledons to the axis. The coty-EPA in germinating jojoba cotyledons. Assays were ledons of the etiolated seedlings were never completely emptiedofgerminatingjojoba cotyledons as described under of seed proteins through 50 days postimbibition (Fig. 3). The lipidds" at (A) pH 4 (0), pH 5 (U); and (B) pH 6 (0), pH reserves in the cotyledons were also not completely depleted3ermination is expressed as days postimbibition or during germination in a growth chamber (19). This may not beseeds (day 0). Duplicate assays were conducted at the case in field conditions where root growth is less restricted andconstant throughout germination and results were up to 60 cm of tap root may develop before notable shoot growth

le. A/h.seed = activity per cotyledon pair. has occurred (13, 34).

JOJOBA PROTEASES 1341

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Plant Physiol. Vol. 68, 1981

Table I. Mixing Experiments to Determine Endogenous Inhibitor ActivityAgainst Proteolysis During Germination

Equal volumes ofextracts from 3- and 37-day postimbibition cotyledonswere mixed and then assayed for proteolytic activity. Expected additiveactivity is the calculated value showing proteolytic activity if no inhibitorwere present in either extract. Measured activity is the actual proteolyticactivity in the experimental, mixed extract assay. Activity per cotyledonpair is reported as A/h * seed.

Expected Ad- Measured Ac- Loss of Ac-Protease Assay ditive Activity tivity tivity

A/R.seed SAPA 86.2 110.7 0CPA 87.8 18.0 80EPA, pH 4 0.06 0.04 aEPA, pH 5 0.4 0.6 0EPA, pH 6 0.30 0.08 74EPA, pH 7 0.06 0.17 0EPA, pH 8 0.20 0.28 0

a , No assessment made due to low activities.

Table II. Effect of Chemical Inhibitor Treatments on CotyledonaryEndopeptidase Activity

Treatments were added to assay mixtures containing gel-filtered coty-ledonary extract from days during germination showing peak activity. Themixture was preincubated at 5°C for 12 h, then warmed to 45°C andassayed as described. Per cent inhibition was determined from comparisonwith control assays containing no chemical treatment.

ProesIoConcen- EndopeptidaseProtease Inhibitor taino

-tration ofTreatments Inhibitor pH 4 pH 5 pH 6 pH 7 pH 8

mM %

Control 0 100 100 100 100 100pCMB8 1.0 0 44 1,900pCMB + Cys 1.0 90 100 0NEM 3.0 65 0 0 0 1,400ME 2.5 67 191 300 109 67PMSF 1.0 78 109 117 200 0EDTA 1.0 157 121 300 114 1,8001,10-Phenanthroline 1.0 0 80 218 188 1,700MgCl2 1.0 82 136 183 70 0

apCMB was not soluble or active below pH 6.

Endogenous Enzyme inhibitors of Jojoba Peptidases. Mixingstudies were conducted to determine if endogenous substances inthe mature jojoba seed were responsible for modification of in situproteolytic activity during germination. Gel-filtered extract from3-day postimbibition cotyledons was mixed with similarly treatedextract from 37-day cotyledons and EPA, APA, and CPA were

assayed (Table I). The expected additive activity is the calculatedvalue ofproteolytic activity ifno inhibitors were present in extractsfrom either day during germination. Measured activity is theexperimentally determined activity of the mixed extract assay.The EPA at pH 6 of the mixed extract was inhibited 70% from theexpected activity. No inhibition of other EPA was detected. CPAfrom extract of 3-day postimbibition cotyledons was inhibited 80oby the extracts from 37-day postimbibition cotyledons. No inhi-bition ofAPA was detected in germinating jojoba cotyledons.

Chemical Inhibitor Studies to Characterize the Proteases. Theeffects of the chemical inhibitor treatments on EPA are shown inTable II. The Azocoll digesting activity (EPA) present in the crudeextract at pH 4 was partially inhibited by NEM, ME, MgCl2, andPMSF. EPA atpH 4 was stimulated by EDTA but totally inhibitedby phenanthroline. Complete inhibition of activity by phenan-throline may indicate the presence of the sulfhydryl enzyme since

this chelator has been shown to oxidize -SH groups of enzymes(33). EPA at pH 5, 6, and 7 showed inhibition by sulfhydrylenzyme inhibitors; EPA at pH 5 was inhibited by NEM and EPAat pH 6 and 7 were inhibited by NEM andpCMB. The inhibitionby pCMB was reversible for EPA at pH 6 and 7 by the additionof cysteine. Inhibition by NEM could not be reversed (see also 4).EPA at pH 5 and 6 (but not at pH 7) were greatly enhanced bythe addition of ME, further indicating activity due to sulfhydrylenzymes. EPA at pH 6 and 7 were differentially increased by thepresence of metal ion chelators EDTA and phenanthroline but, atpH 5, phenanthroline inhibited activity by possibly oxidizing-SH groups at the active site. No metal ion requirement for thesulfhydryl enzymes was found since enzymic activity increased inthe presence of chelators. No clear pattern of requirement forMg2+ by the sulfhydryl enzymes is shown by the data, but Mg2+did increase activity at pH 6 and, to a lesser degree, at pH 5 whileslightly decreasing activity at pH 7. Inhibition of sulfhydryl en-zymes by heavy metal ions may be caused by the formation ofmercaptides with the -SH group (4). The Azocoll-digesting ac-tivity at pH 8 but not pH 7 was completely inhibited by the serineinhibitor PMSF, and by cysteine, and Mg ions (Table II). Inhibi-tion of activity by Mg2+ could be totally reversed by the additionof EDTA (data not shown). Alkaline EPA activity in vitro wasincreased 14- to 19-fold in the presence of the sulfhydryl inhibitorsand chelators (Table II).The effects of inhibitor treatments on APA are shown in Table

III. The APA was totally inhibited bypCMB and inhibited slightlyby NEM. The inhibition of pCMB was totally reversed withcysteine. Complete inhibition of some sulfhydryl enzymes byNEM may require higher concentrations of the inhibitor than

Table III. Effects of Chemical Protease Inhibitor Treatments onCotyledonary Aminopeptidase and Carboxypeptidase Activities

Experiments were conducted as in Table II.

Concentra-Protease Inhibitor Treatment tion of In- APA CPA

hibitor

mm ActivityControl 0 100 100pCMBa 1.0 0pCMB + Cys 1.0 100NEM 3.0 86 79ME 2.5 85 78PMSF 1.0 85 5EDTA 1.0 121 1611,10-Phenanthroline 1.0 114 148MgC12 1.0 47 133

apCMB was not soluble or active in the assay for CPA at pH 5.0.

Table IV. Inhibition of Trypsin, Chymotrypsin, Pepsin, and FungalProtease by Jojoba Seed Extracts

Proteinases of equal specific activities on Azocoll were added to assaymixtures containing 0.4 ml gel-filtered jojoba cotyledonary extracts, thenpreincubated at room temperature for 15 min. Azocoll assays were con-

ducted as described. Inhibition of protease activity was determined bycomparison with control activities containing no jojoba seed extract.

% Inhibition by 0.4 mlProteinase Seed Extract

Bovine trypsin 100Bovine chymotrypsin 92Pepsin 64Fungal ob

a Protease from A. saotorib No inhibition of fimgal proteinase with any volume of extract was

observed.

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Plant Physiol. Vol. 68, 1981

Table V. Inhibition of Bovine Trypsin Activity by Jojoba Seed ExtractsTrypsin (2.5 ytg/ml) activity was assayed in the presence of gel-fitered

jojoba seed extracts prepared as described under "Materials and Methods."Mixtures of trypsin (2.0 ml) plus extract in various amounts were prein-cubated at room temperature for 15 min then assayed with Azocoll.Inhibition was determined from activities of control assays containing nojojoba seed extract. The addition of 3.4 Lg commercial soybean trypsininhibitor caused complete inhibition of trypsin activity (5 ytg trypsin/assay).

Source of Extract Wt Causing 100% Inhibi-tion"mg

Jojoba cotyledons, day 6 48Jojoba cotyledons, day 37 192Jojoba seed mealb 110Jojoba seed albuminb 0.19Jojoba seed globulnnb NDcJojoba liquid waxb ND

aAmount of dry material in the extract added to the assay mixture.b Extracted from mature, dry seed.c Not detected or no inhibition with any amount of material.

used in this study (4). The APA was also inhibited by MgCl2 andinhibition could be reversed with EDTA (data not shown). NoME activation of APA was found. There was apparently norequirement for a heavy metal ion since activity was insensitive tochelators.

Results of inhibitor treatments on CPA are shown in Table III.Inhibition of activity was caused by the seine enzyme inhibitorPMSF. Optimum proteolytic activity did not require the additionof metal ions (activity was increased with EDTA) or ME.

Influence of Endogenous Jojoba Seed Inhibitors on Proteinasesfrom Other Organisms. Azocoll assays were conducted to deter-mine if extracts of germinating jojoba seeds would inhibit thebovine proteinase enzymes trypsin, alpha-chymotrypsin, porcinepepsin, and the protease from the fungus A. saotoi (Table IV).The trypsin inhibitor activity in the jojoba extract was greaterthan the chymotrypsin or pepsin inhibitor activity assayed undersimilar conditions. No amount ofjojoba seed extract inhibited theactivity of the fungal protease (Table IV).The trypsin inhibitor activity was also found in buffered extracts

prepared from commercial processed jojoba seed meal. This activ-ity was eluted with the void volume during gel filtration (SephadexG-25, data not shown) and was localized in the albumin fractionofjojoba seed protein (Table V). No trypsin inhibitor activity wasexhibited by the globulin fraction or the liquid wax (Table V).Trypsin inhibition by extracts from jojoba seed was found to belost by gently boiling (2 min) and then cooling of the extractbefore addition to the assay mixture (data not shown). Thus, theabove cumulative evidence suggests the trypsin inhibitor ofjojobaseeds is a protein.During germination, the trypsin inhibitor activity in the coty-

ledons decreased 4-fold from imbibition to 37 days postimbibition(Table V). A cotyledon pair (1.1 g) from 6-day-old seedlingscontained trypsin inhibitor activity equal to ,ug purified soybeantrypsin inhibitor (Worthington code SI).

DISCUSSION

The data indicate that relatively low levels of-SH and alkalineEPA, APA, and later CPA were involved in the early loss ofprotein from the germinating jojoba cotyledons. Later duringgermination the loss of protein from the cotyledons was probablyassociated with the rapid increase in -SH EPA at pH 4 and 5and later at pH 6. Mixing experiments indicate that the rise inEPA at pH 6 and in CPA later in germination may be the result

of lower levels of the substances which inhibited these activitiesearly in germination. A similar situation occurs in wheat (23) andan endogenous inhibitor of EPA at pH 6 has been reported inmung beans (3). EPA at pH 7 and 8 did not appear to play amajor role in reserve protein catabolism in jojoba seeds.

Studies by other workers (19, 31) suggest that metabolism ingerminating jojoba and castor bean may be similar. In addition,studies on castor bean (31), corn endosperm (12), bean (11) andpea (20) show a similar pattern ofAPA and CPA to that in jojobaduring germination; early in germination APA is high and CPAlow, while later there is a rise in CPA and -SH EPA concurrentwith rapid depletion ofprotein. In pea seedlings, a phenanthroline-insensitive enzyme is the major APA and probably functions inthe general turnover of cellular proteins (8). The major jojobaAPA is also phenanthroline-insensitive and is also probably moredirectly involved in cellular protein tumover than reserve proteindegradation. Increases of -SH EPA may be a prerequisite forsubsequent protein degradation during germination. Peptidesformed as a result of EPA in the cotyledons could be degraded toindividual amino acids by CPA which remained at a relativelyconstant level through later stages of germination in jojoba. Asimilar situation has been suggested in other seeds (1, 2, 7, 33).The developmental data given above show distinctly different

temporal curves of pH-dependent proteolytic activity (EPA, APA,CPA) following imbibition by jojoba seeds. Thus, we suggest thecoordinated oncogenetical and changing functional operation ofmultiple forms of proteolytic activity in the germinating cotyle-dons. Additional evidence for the different forms of these enzymeactivities was provided by the chemical treatment studies (TableI, II). EPA at pH 4, 5, 6, and perhaps at 7 may be due to multipleforms of sulfhydryl protease activity. This hypothesis is based onthe generally similar, but individually different, responses tosulfhydryl inhibitors and activators, PMSF, chelators, and Mg2".Other workers have classified several plant endopeptidases assulfhydryl enzymes exhibiting acid pH optima (6, 7, 12, 31, 33).Azocoll digestion at pH 8 was probably primarily due to serineprotease activity. APA was a sulihydryl and CPA a serine proteasein the jojoba cotyledons. Most plant APA have been shown to bedue to a sulfliydryl enzyme with a pH optimum near 7.0 (10, 12,26, 31, 33) and CPA reported to be due to a serine protease mostactive near pH 5.0 (6, 7, 12, 14, 22). Any possible role in limitedproteolysis by an individual protease is being studied in ourlaboratory.

Multiple forms of endoproteolytic activity have been reportedin bean seedlings (9, 11, 26, 31), barley (6, 17), pea embryo axes(20), and castor bean (31, 33). Identification of the individualactivities is typically based on pH, substrate specificity, or occa-sionally, inhibitor studies. It is prudent to use more than onemethod of identification in developmental enzyme studies. More-over, most developmental studies on proteolytic activity report aseries of enzyme assays performed at only one pH value duringthe entire course of the study. We suggest that conducting assaysat a range of pH values may guard against obtaining possiblemisleading results concerning temporal shifts in enzymic activity.We recognize that the substrates used in the jojoba seed assays

are not the physiological substrates and data obtained from theseassays indicating the roles and mechanisms of jojoba seed pro-teases may not entirely reflect the in vivo situation (see also 31).However, artificial substrates have been used extensively to char-acterize various proteases (1, 33 and see under "Materials andMethods"). Moreover, it was not possible to assay the jojobaproteases with radioactive protein (2) because no developing seedswere available to prepare the substrate (see also 21); flowering injojoba takes 3 to 6 years after seedling establishment in the wild(13, 34). With proper controls and care taken in the procedures,the Azocoll assays were reliable and results were reproducible.The presence of animal digestive proteinase inhibitors in many

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Plant Physiol. Vol. 68, 1981

seeds has long been recognized but not well understood (15). Ryan(26) has proposed that these inhibitors may act as storage proteins,as a mask for preexisting plant proteases in the seed or to protectthe seed from animal and microbial attack. The presence ofanimaldigestive-proteinase inhibitors in jojoba seed extracts and mealmay have important implications in the use of jojoba meal forfeed and could clarify conflicting and unexplained data reportedfrom studies of the toxic effect of the meal in animal feedingexperiments (13, 32 and references therein). Since the meal maybe heat-treated to inactivate other toxins (32), and in our studies,heat inactivated the trypsin inhibitor, its presence in the meal maynot be a major obstacle to its commercial use.

Jojoba seeds may contain an endogenous inhibitor(s) of alkalineEPA which apparently required an -SH group to function. Twoobservations support this possibility. First, the decreased alkalineEPA in the presence ofME and cysteine which may have activatedthe -SH requiring endogenous inhibitor(s) and second, thegreatly increased alkaline EPA in the presence of chemical sulfhy-dryl inhibitors which may have inactivated the -SH requiringendogenous inhibitor(s) (Table II). A similar inactivation of an-SH inhibitor has been reported in animal tissues (16). In jojoba,inhibiting prococious EPA at pH 8 during germination could beimportant for seeds growing in alkaline soils of the SonoranDesert.

Acknowledgments-The authors thank Dr. W. Heim and Dr. R. Taber of TheColorado College for critical discussions concerning this research and Tammy Sageand Carol Lovejoy for technical assistance.

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1344 SAMAC AND STOREY

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