Characterization of the Major Protease Involved the ... Chromatography ... The gel, shown in Figure 1, was analyzed for the degradationof,B-conglycinin subunitsandfortheappearance

Plant Physiol. (1992) 99, 725-7330032-0889/92/99/0725/09/$01 .00/0

Received for publication October 21, 1991Accepted January 5, 1992

Characterization of the Major Protease Involved in theSoybean ,3-Conglycinin Storage Protein Mobilization1

Xiaoqun Qi, Karl A. Wilson, and Anna L. Tan-Wilson*

Department of Biological Sciences, State University of New York at Binghamton, P.O. Box 6000,Binghamton, New York 13902-6000

ABSTRACT

Protease Cl, the protease responsible for the initial degrada-tion of the a' and a subunits of the soybean ,-conglycinin storageprotein (Glycine max [L.] Merrill), has been purified. The enzymewas found by sodium dodecyl sulfate-polyacrylamide gel electro-phoresis to have a molecular weight of 70,000 and a pH optimumof 3.5 to 4.5. Susceptibility to protease inhibitors indicates thatprotease Cl is a serine protease. Study of the proteolytic inter-mediates generated suggests that the cleavage of the a' and asubunits of 8-conglycinin by protease C1 results in intermediatesthat are 1 or 2 kilodaltons smaller than the native a' and asubunits. Following that, a succession of intermediates exhibitingmolecular masses of 70.0 and 58.0 kilodaltons, then 63.0, 61.0,55.0, and 53.5 kilodaltons, are observed. A 50.0- and a 48.0-kilodalton intermediate are the final products of protease Claction. Comparison of these intermediates with the prominentanti-,8-conglycinin cross-reacting bands that increase during thefirst few days of germination and early growth show that proteaseCl plays an important physiological role, but not an exclusiveone, in the living plant.

A major metabolic event in the germinating seed is thehydrolysis of the seed protein reserves to provide the growingseedling with the nutrients necessary before photosynthesis isestablished. There are two major storage proteins in thesoybean (Glycine max [L.] Merrill) seed, glycinin and fl-conglycinin, which together make up 70% of the seed proteinreserves by dry weight. Glycinin consists of six nonidenticalsubunits. Each subunit has one acidic chain and one basicchain linked by a single disulfide bond (16). ,B-Conglycinin isa glycoprotein that is composed of three noncovalently asso-ciated subunits, a', a, and # (23, 24), with mol wts of 76,000,66,000, and 47,500, respectively. During germination andearly growth, these storage proteins are degraded by proteol-ysis. The predominant pattern is one of limited proteolyticcleavage by proteases specific for the reserve protein, followedby more rapid proteolysis by less specific proteases (15, 19).

Several soybean proteases have been described. Theseinclude six proteolytic enzymes from ungerminated seed sep-arated by anion-exchange chromatography (26), two carbox-ypeptidases from germinating soybeans (10), two endopepti-dases, one exhibiting an acidic pH optimum and the other a

Supported by National Science Foundation grants PCM 8301202and DCB 9017420.

basic pH optimum (2), and a trypsin-like protease (17). Exceptin the case of the acidic and the basic endopeptidases, whereendogenous substrate was also tested, only exogenous orsynthetic substrates were used to determine the proteolyticenzyme activities. At times this strategy leads to the identifi-cation of proteases that may be physiologically irrelevant.This was aptly demonstrated in a recent report (7) in whichthe authors showed the discrepancy when using synthetic,exogenous, and endogenous substrates. In past reports, work-ing with small seed proteins such as the trypsin inhibitors assubstrates, we have successfully found proteases that not onlydegraded the native substrate proteins but also generated thesame products of limited proteolytic cleavage found in theseedling (6, 12).

In this study, our objective was to discover and purify themain enzyme responsible for the in vivo proteolytic cleavageof one of the storage proteins, and then to delineate thepathway of specific limited proteolysis that mobilizes thestorage protein for further degradation. We have establishedassay conditions for the enzymes catalyzing the degradationof the a' and a subunits of 3-conglycinin using the native f-conglycinin, not an exogenous protein, as the substrate.A previous study on the pattern of the degradation of f-

conglycinin in vivo (27) showed that of the three subunits of3-conglycinin, the a' and a subunits are more rapidly de-graded upon seed imbibition. Proteolysis is accompanied bythe accumulation of a smaller discrete 13-conglycinin cross-reacting band exhibiting a mol wt of 51,200 on SDS-PAGE.We have recently been able to resolve the band and find threeproteolytic intermediates of mol wt 50,000, 51,800, and53,500. The A subunit was also shown to be degraded but ata slower rate (27).

In this paper, we describe the purification and characteriza-tion of a proteolytic enzyme (protease Cl), which is respon-sible for the early degradation of the a' and a subunits of3-conglycinin in the living plant. This protease acts on B-

conglycinin to produce a series of 11 proteolytic intermediatesexactly like those found in seedling cotyledons. The finalproducts of a' and a subunit degradation are the two prote-olytic intermediates of mol wt 50,000 and 48,000. By com-paring the time course of appearance and disappearance ofintermediates upon proteolysis of f-conglycinin by purifiedprotease C1, we are able to propose a hypothetical scheme ofthe main pathway of limited proteolytic cleavage of f-congly-cinin. Study of this enzyme is especially crucial because itcatalyzes one of the very first reactions in the mobilization ofseed reserves in the soybean.

725

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Plant Physiol. Vol. 99, 1992

MATERIALS AND METHODS

Plant Material and Germination of Seeds

Soybean seeds (Glycine max [L.] Merrill, cv Amsoy 71)were obtained from May Seed and Nursery Company (Shen-andoah, IA). Seeds were planted at equal depth covered by 1cm of moist vermiculite and grown in a growth chamber witha 12-h light and 12-h dark photoperiod. The temperature ofthe growth chamber was set at 25°C in the light and 20°C inthe dark. Plants were watered with distilled water, and grownfor a period of up to 12 d. Cotyledons were harvested atspecified days of growth (reckoned relative to the beginningofimbibition), rinsed in distilled water, blotted dry, and storedat -80°C until needed.

Preparation of Extracts

Cotyledons were homogenized in a Waring blender in ice-cold 50 mM NaPi + 2.0 mM DTT, pH 7.0, using 7 mL/g freshweight. The homogenate was centrifuged twice at 19,000g for25 min at 4°C. The supernatant was then collected and frozenin aliquots at -20°C.

Assay of fi-Conglycinin-Degrading Activity

,B-Conglycinin-degrading activity was assayed using ,B-con-glycinin purified (27) by zonal isoelectric precipitation chro-matography. The substrate mixture consisted of 10 ML ,B-conglycinin (17.3 gg), 2 uL of antibiotic mixture (2 mg/mLkanamycin + 0.1 mg/mL amphotericin B), 4 ,L of 4 mMDTT, and 24 ML offour-times concentrated McIlvaine citrate/phosphate buffer (13). To this was added 5 uL of the sampleof interest. The reaction was incubated at 37°C for the desiredtime, then terminated by adding 45 uL of 2 x Laemmli SDS-PAGE treatment buffer (11), heating at 95°C for 10 min, andfreezing at -20°C.For each sample, two identical reaction mixtures were

prepared. One was terminated immediately to serve as a zero-hour control; the other was incubated for the appropriate timeand then stopped as described above. The reaction mixtureswere then subjected to SDS-PAGE as described by Laemmli(1 1). The gels were stained for protein with Coomassie blueG (Serva Chemical Co.) (0.1% [w/v] in 50% [v/v] methanol+ 10% [v/v] acetic acid), and destained in 12% (v/v) 2-propanol + 10% (v/v) acetic acid. The gels were then scannedusing a Hoefer model GS300 scanning densitometer or anLKB laser scanning densitometer, followed by planimetry tomeasure the peak areas of the individual subunits of 13-conglycinin. Results were expressed as the percentage disap-pearance of each subunit, which equals 100 x [((zero-hourpeak area) - (reaction-hour peak area))/zero-hour peak area].One enzyme unit is defined as the amount ofenzyme neededto digest 1 MAg of a' or a subunit of f3-conglycinin in 24 h at37°C. To transform the percentage of disappearance into Mugof subunits degraded, we used an extinction coefficient of ,3-conglycinin, E2% = 4.16 (24), to calculate the amount ofinitial ,B-conglycinin protein, and relied on relative stainingintensities of the subunits on SDS-PAGE to calculate theconcentration of each subunit. The assumption that the threesubunits have similar staining intensities is supported by our

finding that plots of staining intensity versus amount ofprotein are parallel for the a', a, and ,B subunits.

SDS-PAGE and Western Blotting

Cotyledon extracts were examined by SDS-PAGE by themethod of Laemmli (11). For immunodetection, the sepa-rated peptides were transferred to Immobilon PVDF mem-brane (Millipore) and immunostained using secondary anti-body conjugated to horseradish peroxidase as describedpreviously (27).

Enzyme Purification

Extraction

Soybean cotyledons (220 g) harvested from day 12 seedlingswere homogenized using 1 L of ice-cold 50 mM NaPi + 10mM 1.-MET2, pH 7.0. All subsequent steps were carried outat 0 to 4°C. The mixture was filtered through four layers ofcheesecloth, centrifuged as previously described, and the su-pernatant collected. The extract was then titrated to pH 5.0with acetic acid, dialyzed against 50 mm Na acetate + 10 mM,3-MET, pH 5.0, and centrifuged to remove the precipitatedstorage proteins.

Ion-Exchange Chromatography

The crude extract obtained above was applied to a CM-Trisacryl M column (3 x 60 cm) equilibrated using 50 mMNa acetate + 10 mm fl-MET, pH 5.0. The same buffer wasalso used to elute the loosely bound proteins, and a linear saltgradient (800 mL, 0-0.6 M NaCl in 50 mM Na acetate + 10mM ,B-MET, pH 5.0) was applied to elute the enzymes.Fractions of 5 mL were collected at 60 mL/h, and enzymeactivities determined. Fractions 284 to 305 were pooled andsubjected to further purification.

This pool was dialyzed against 50 mM NaPi + 10 mM p-MET, pH 6.5, and applied to a DEAE-Trisacryl M column(2.5 x 45.5 cm) equilibrated to the same buffer. After elutingthe loosely bound proteins with the starting buffer, the columnwas eluted with a linear salt gradient (500 mL, 0-0.6 M NaClin 50 mM NaPi + 10 mM fl-MET, pH 6.5). Elution was at 47mL/h with 5-mL fractions collected. The fractions having 1l-conglycinin-degrading activity were pooled and concentratedby ultrafiltration using an Amicon YM 2 membrane.

Sephadex G-75 Gel Filtration Chromatography

The concentrated pool from the DEAE-Trisacryl chroma-tography was applied to a 1.5 x 90 cm column of SephadexG-75 (Pharmacia) equilibrated to 50 mM NaPi + 10 mm ,B-MET, pH 6.5. The column was eluted with the same bufferat 21 mL/h, with 1.5 mL collected. The fractions were assayedfor activity, and examined for purity by PAGE and SDS-PAGE (11) followed by silver staining (30).

2Abbreviations: ,B-MET, ,B-mercaptoethanol; BBSTI, Bowman-Birk soybean trypsin inhibitor; KSTI, Kunitz soybean trypsininhibitor.

726 Ql ET AL.



SOYBEAN 1-CONGLYCININ-DEGRADING ENZYME

Inhibition Studies

To study the effect of protease inhibitory reagents on the13-conglycinin-degrading activity, purified enzyme was prein-cubated with the appropriate concentrations of various re-

agents at 0°C for 2 h. The concentrations of inhibitors in thepreincubation mixtures were chosen as previously described(29). The protein trypsin inhibitors used (BBSTI and KSTI)were purified as described by Tan-Wilson and Wilson (21).Inhibitors were dissolved in different solvents: PMSF in 2-propanol; E-64 in 6% DMSO; pepstatin, leupeptin, Na io-doacetate, EDTA, KSTI, and BBSTI in water. Control incu-bations with the addition of only these solvents to the enzymesolution were also performed. After preincubation, the re-

maining proteolytic activity was assayed as described above,with the exception that DTT was not added.

RESULTS

Proteolytic Intermediates Resulting from the Degradationof 13-Conglycinin in Vivo

Soybeans were planted and harvested at specified daysduring the first 12 d after imbibition. Extracts prepared byhomogenizing the cotyledons in cold 50 mm NaPi + 2.0 mMDTT, pH 7.0, were examined on SDS-PAGE in the Laemmlisystem. The gel, shown in Figure 1, was analyzed for thedegradation of ,B-conglycinin subunits and for the appearanceof proteolytic intermediates. Of the three subunits of 13-con-

glycinin, the subunit was found to be degraded at the slowestrate, persisting up to day 6. The a' and a subunits, on theother hand, were found to be rapidly degraded during earlygrowth, having largely disappeared by day 4. In their placeappear a set of polypeptides of mol wts lower than those of

R1 2 3 4 5 6 7 8

92-

66"

45-

31'

a'

58

- 53.5451.8

it50

45

Figure 1. SDS-PAGE of extracts from cotyledons harvested ondifferent days of early growth. Extracts of cotyledons were preparedand electrophoresis carried out in the Laemmli system on 10% gelsas described in 'Materials and Methods." Samples loaded into eachwell contained extract equivalent to 0.01 cotyledon. The locations ofthe a', a, and ,3 subunits of f-conglycinin are indicated to the right ofthe gel, as are the intermediates generated from the degradation ofthe a' and a subunits. Lanes 1 to 7, extracts from days 1, 2, 3, 4, 5,6, and 8, respectively. Lane 8 is the same as lane 3, repeated toclarify labeling of the intermediates. The positions of molecular massstandards (not shown in the figure) are indicated to the left of the gel.

1 2 3 4 5 6 7 8 kD

58

53.551.8-50

45

-'116--97

-"66

-"45

,,"29

Figure 2. Western blot of the ,B-conglycinin degradation intermedi-ates. SDS-PAGE was performed as in Figure 1, except in a 7.5% gelto improve resolution. The resolved polypeptides were electrotrans-ferred onto Immobilon PVDF membrane, and immunostained usinganti-13-conglycinin-specific antibody and goat anti-rabbit immunoglob-ulin G-horseradish peroxidase. The a', a, and d subunits are labeledto the left of the blot, as are six of the degradation intermediates.Lanes 1 to 8 correspond to extracts from days 1, 2, 3, 4, 5, 6, 8, and10, respectively. The position of molecular mass standards run onthe same gel are indicated to the right of the blot.

the a' and a subunits, but larger than the p subunit. On awestern blot (Fig. 2), these newly appearing proteins werefound to cross-react with anti-fl-conglycinin antibody, whichsuggests that they are proteolytic degradation products of thea' and a subunits of fl-conglycinin.The intensities of the Coomassie blue-stained bands were

compared by scanning densitometry and the results are pre-sented in Figure 3. Panel A shows the degradation of thenative subunits of ,-conglycinin: more rapid degradation forthe a' and a subunits, slower for the : subunit. Panels B andC show the generation and subsequent disappearance of theproteolytic intermediates. Those intermediates that followsimilar temporal patterns of appearance and disappearanceare grouped together in panel B. The 55.0-, 53.5-, 51.8-,50.0-, and 45.0-kD intermediates appear between day 1 andday 2, peak at day 3, are sustained until day 5, and are thendegraded rapidly after that. Although the 45.0-kD intermedi-ate has a lower molecular mass than the 13 subunit of 1-conglycinin, the following experimental observation suggeststhat it is a degradation product of either the a' or a subunitof 1-conglycinin or of both. When crude extracts containingthe ,B-conglycinin storage protein were separated by SDS-PAGE, electroblotted onto nitrocellulose membrane, andthen stained with our anti-#-conglycinin (which binds to thea' and a subunits more strongly than to the p subunit), the45.0-kD intermediate was found to stain more strongly thanthe 13 subunit, even when the level ofthe 45.0-kD intermediatewas much lower than that of the 13 subunit as observed withthe Coomassie blue protein stain.

Panel C shows that two other prominent intermediates(58.0 and 48.0 kD) do not follow the pattern observed inpanel B. The 58.0-kD intermediate was found to be presentin the day 1 extract. Levels decrease as early as day 2 afterimbibition, until the intermediate is barely discernible by day4. The 48.0-kD intermediate appears only by day 4, reaches

727




2 0

1.0

a-

OOR*

0.6

0.3

0.4

0-2

0 2 4 6 8 10

DAYS AFTER IMBIBITIONFigure 3. Temporal variation of ,B-conglycinin polypeptides and thein vivo intermediates of a' and a subunit degradation. The SDS-PAGE pictured in Figure 1 was quantified by scanning densitometryand planimetry. The areas of the peaks corresponding to specificpolypeptides were plotted versus time after seed imbibition. A, Thea', a, and : subunits; B, the 55.0-, 53.5-, 51.8-, 50.0-, and 45.0-kDintermediates; C, the 58.0- and 48.0-kD intermediates.

peak levels at day 6, and is then degraded. Our finding of thetemporal profiles shown in Figure 3C, apparently unrelatedto those shown in panel B, suggests that the a' and a subunitsare subjected to more than one pathway for initial proteolyticdegradation.

,8-Conglycinin-Degrading Activity during Early Growth

Assays of proteolytic enzyme activity catalyzing the degra-dation of the a' and a subunits of 13-conglycinin were con-

ducted on extracts of seedling cotyledons harvested duringthe first 12 d of growth. Because extracts made from cotyle-dons from the early days of growth contained large amountsof,B-conglycinin, whereas those from later days containedlittle or no ,3-conglycinin, f3-conglycinin was added so that all

incubation mixtures would have equal amounts of f,-congly-cinin. The results indicate a steady increase in a subunit-degrading activity up to day 12. No activity was observed inthe extract of ungerminated seeds. The enzyme activity (ex-pressed as mg of a subunit degraded/24 h - cotyledon) was 0.6to 0.8 for days 1 to 3, 3.3 for day 6, 7.5 for day 8, and 20 forday 12. As shown in Table I, the intermediates generated inthe in vitro degradation of the a' and a subunits of ,B-conglycinin in the assay with day 12 extract were the same asmany of those intermediates observed in vivo, and thosegenerated upon incubation of,-conglycinin with day 1 and

day 6 cotyledon extracts. This indicates that, although thenative subunits of,-conglycinin are largely degraded by day12 (Fig. 3), the enzyme catalyzing ,B-conglycinin degradationin the earlier days is still present at day 12.

Purification of ,8-Conglycinin-Degrading Enzyme

The cotyledons from day 12 of growth were used as thestarting material for the purification of the a' and a subunit-degrading activity. This choice capitalized on the fact that thelevel of enzyme activity is high in this tissue, whereas storageprotein levels are very low. Specific details of the purificationprocedures are given in "Materials and Methods." The chro-matography of the crude enzyme extract on CM-Trisacryl Mis shown in Figure 4. Three peaks of activity degrading the a'and a subunits of 3-conglycinin were observed. Peaks 2 and3 were found to degrade the a' and a subunits mainly to the45.0-kD intermediate, although the 55.0-, 53.5-, and 50.0-kDintermediates were also observed. In contrast, only peak 1

was found to generate the same distinct intermediate observedin vivo. Peak 1 was thus further purified.Chromatography of the material from peak 1 on DEAE-

1 2 Trisacryl M resulted in a single major peak of activity, elutingat approximately 0.02 M NaCl, which produced the same

Table I. Comparison of the Intermediates of in Vivo and in Vitrof3-Conglycinin Degradation

Intermediates are identified by their molecular mass in kD.In Vitro

In Vivoa Day 1 Day 6 Day 12 Protease

extractb extracP extractc Cl1

74.0 74.070.0 70.0 70.065.0 65.0 65.0

63.061.059.0

58.0 58.0 58.0 58.0 58.055.0 55.0 55.0 55.053.5 53.5 53.5 53.5 53.551.8 51.850.0 50.0 50.0 50.0 50.048.0 48.045.0 45.0 45.0

a Internediates observed dunng growth over 1 to 12 d. b Inter-mediates generated during autolysis of day 1 extract at pH 4 for24 h. c Intermediates generated during incubation of f3-conglycininwith either day 6 or day 12 extract, or purified protease Cl.

728 Ql ET AL.



SOYBEAN ,B-CONGLYCININ-DEGRADING ENZYME

A B

920

66flz 0z.

FRACTIONS

Figure 4. Ion-exchange chromatography of 13-conglycinin-degradingactivity on CM-Trisacryl M. The crude extract from 220 g of day 12cotyledons was dialyzed against 50 mm Na acetate + 10 mm ,B-MET,pH 5.0, and applied to a 3 x 60 cm column of CM-Trisacryl equili-brated to the same buffer. The column was eluted at 60 mL/h with a1 600-mL linear gradient (0-0.5 M) of NaCI in the buffer. Fractions of5 mL were collected. Fractions were assayed for activity catalyzingthe degradation of a' and a subunits, expressed in the percentageof the initial substrate (17 Mig ,-conglycinin) degraded in 24 h by 15,ML of column fraction. -, A280; 0, enzyme activity; *, M NaCI. Thethree peaks of enzyme activity are labeled at the top of the figure.Fractions 284 to 304, corresponding to peak 1, were pooled asindicated.

intermediates ofdegradation as observed in vivo. This materialwas concentrated and chromatographed on Sephadex G-75.Again, a single peak of activity degrading the a' and a

subunits and producing the same intermediates was found.This material was pooled, concentrated by ultrafiltration, andserved as the purified enzyme for further studies.The results of this purification are summarized in Table II.

The similar levels of recovery and purification of the proteo-lytic activities directed against the a' and a subunits indicatethat the same enzyme is responsible for the degradation ofboth of these subunits. The largest loss in a' and a subunit-degrading activity occurred in the CM-Trisacryl chromatog-raphy. The material applied to this column contained approx-imately 70% of the total a' and a subunit-degrading activityin the extract. However, only 8% ofthis activity was recovered

3e_

Figure 5. Electrophoresis of purified protease Cl. A, SDS-PAGEshowing, from left to right, molecular mass standards (expressed inkD) and protease Cl (indicated by the arrow). B, NondenaturingPAGE at pH 8.3, with the position of protease Cl indicated by thearrow. Both gels were silver stained.

in the pool of peak 1. This is primarily because the majorityof a' and a subunit-degrading activity is found in peaks 2and 3, which, however, do not produce the large number ofphysiologically relevant proteolytic intermediates shown inFigure 3.The final protease preparation was judged to be 92% pure

by SDS-PAGE (Fig. 5A). Contaminants were lower mol wtproteins that may be autolysis products. NondenaturingPAGE at pH 8.3 exhibited a single protein band (Fig. 5B).Because this enzyme catalyzes the initiating proteolytic cleav-age steps for fl-conglycinin, we will refer to it as protease C 1.

Mol Wt

The single major band observed in SDS-PAGE (Fig. 5A)indicates a mol wt of 70,000 for protease C 1.

Table II. Purification of Protease C1

Activity Specific Recovery PurificationStep Protein Activity

a ax a at a a a a

A280unitSa UnitSb X 10-4 unitsIA280unit % -foldx lo-3

ExtracP 11,818 1,120 1,650 1 1 100 100 1 1CM-Trisacryl 53 88 142 18 28 8 9 20 20DEAE-Trisacryl 16 65 77 41 48 6 5 46 34Sephadex G-75 0.5 40 57 800 1,140 4 3 889 814

a One A2W unit of protein refers to that amount of protein that gives an absorbance of 1.000 at 280nm with a 1 -cm light path when dissolved in 1 mL of solvent. b One unit of enzyme activity causesthe disappearance of 1 Ag of a' or a subunit at 370C in 24 h. c Prior to acidification to pH 5.0.

0cocmj

729

doom




loo - Mechanistic Class of Protease ClThe effect of various reagents on the pure enzyme was

.> 1l > examined as described in "Materials and Methods." The

.> 1l \ \ purified enzyme did not require DTT to be present for fullO: / t t | activity. Therefore, all assays in this section were performed< in the absence of DTT. The results are shown in Table III..> 50 - / / \ \ Protease Cl is not inhibited by iodoacetate or E-64 (inhibitorsco of cysteine proteases), pepstatin (an inhibitor of aspartic pro-

teases), EDTA (an inhibitor ofmetalloproteases), or leupeptin.Protease C is only inhibited by PMSF, indicating that it is aserine protease.BBSTI and KSTI are endogenous seed proteins known to

0 inhibit some vertebrate serine proteases. These inhibitors have2 3 4 5 6 7 8

previously been shown to be present in the protein bodies ofpH cotyledon cells (8, 9), and thus in the same subcellular com-

Figure 6. The pH dependence of protease Cl activity. p3-Conglycinin partment as the storage proteins. Therefore, we tested the

(17 Ag.bwasn7ncuintedwwithwpurifiedwenzymeww2w7www10w4wA280unbyhbsi ability of these inhibitors to inhibit protease ClI activity. Our

a series of Mcllvaine buffers modified to be equivalent in ionic results indicate that the predominant forms of these two

strength. Activity is expressed as the percentage of disappearance proteins, the Tipform of KSTI and the E form of BBSTI, are

of the a' (U) and a (0) subunits in a 3-h incubation, not inhibitors against protease Cl

pH Optimum

The pH optimum for the action of protease Cl on (3-

conglycinin was determined at constant ionic strength. Theassay procedure described in "Materials and Methods" was

used, with the exception that the stock citrate/phosphatebuffers used were all adjusted to an ionic strength of 1.0 M(5). The results are shown in Figure 6. The enzyme exhibitsan optimum pH of 3.5 to 4.5 in its proteolysis of the a' anda subunits of i3-conglycinin. No activity against the A subunitwas detected at any of the pH values tested. Thus, the ap-

pearance of subunit-degrading activity detected in day 12extracts must be due to another enzyme.

Substrate Specificity

The proteolytic activity of purified protease C l toward eachof the three ,B-conglycinin subunits and toward glycinin (theother major soybean storage protein) were compared. Thespecific activities of protease Cl toward a' and a subunits off-conglycinin are 800 and 1 140 ,ug/24 h .A280 unit ofenzyme,respectively. Here, one A280 unit of protein is defined as theamount that gives an absorbance of 1.000 at 280 nm whendissolved in 1 mL of solvent with a 1-cm lightpath. When theenzyme activity is translated into terms of nmol of substratedigested, protease Cl activity toward the a subunit, 17.3nmol/24 h .A280 unit of enzyme, is 39% higher than that forthe a' subunit (10.5 nmol/24 h-A280 unit). There is very littleactivity toward the subunit of ,B-conglycinin (4 ,g/24 h-A280 unit) or to the acidic chain of glycinin (21 ug/24 h .A280unit). No activity was found toward the basic chain of glyci-nin. We also tested the purified protease Cl for activity againstthe predominant forms of KSTI and BBSTI, proteins that,although present in small amounts in the soybean, are alsothought to serve as storage proteins (22). Protease Cl had noactivity against either ofthe native trypsin inhibitors nor theirnatural initial degradation products.

Time Course of the Protease Cl Degradation of,-Conglycinin

Protease Cl was incubated with f3-conglycinin substrate fordifferent time periods. The band intensities ofthe degradationintermediates were quantified by SDS-PAGE followed byscanning densitometry and planimetry. The results are pre-sented in Figure 7. Panel A demonstrates the time course ofdegradation of the a' and a subunits of ,3-conglycinin, andshows the lack of activity toward the subunit. Severalproteolytic intermediates are observed during the course ofdegradation. Their temporal patterns are presented in panelsB, C, and D. We have grouped together in each panel thoseintermediates that exhibit similar temporal profiles. Panel Bshows the set of intermediates (74.0, 70.0, 65.0, and 58.0 kD)that are formed within a few minutes of incubation time,reach peak levels within 2 h, and are no longer present by 4or 8 h. Panel C shows the set of intermediates (63.0, 61.0,

Table Ill. The Effect of Inhibitory Reagents on the Activity ofProtease C1

% ActivityRemaining

Reagent Concentrationa Towardb

a a

mM

None (control) 100 100Na iodoacetate 5.0 96 97E-64 0.02 92 98PMSF 2.0 45 41Pepstatin 0.02 93 98Leupeptin 0.02 92 96EDTA 5.0 83 92

a Concentration of the inhibitory reagent in the preincubation mix-ture, before addition of substrate, buffer, and antibiofic mixture. b Per-centage of activity toward the indicated ,3-conglycinin subunit relativeto that of the enzyme control (no reagent added).

730 Ql ET AL.



SOYBEAN 3-CONGLYCININ-DEGRADING ENZYME

10

C14

A Bw 0.6C S - 63.0 kD 59.0 kD< 0.6 - 61.0 kD - 50.0 kD

se - 55.0 kD * 48.0 kD

< ~ ~ ~ -0-53.5 kD

0.30.3

C ~~~~~D0 4 8 12 0 4 8 12

TIME (hours)

Figure 7. Time course of ,B-conglycinin degradation in vitro by purifiedprotease Cl. 3-Conglycinin (17 ,ug) was incubated with protease Cl(2.7 x 10-4 A280 units) for the indicated times. The resulting productswere resolved on SDS-PAGE and quantified by densitometry. A, Thea', a, and d subunits. B, The 74.0-, 70.0-, 65.0-, and 58.0-kDintermediates. C, The 63.0-, 61.0-, 55.0-, and 53.5-kD intermediates.D, The 59.0-, 50.0-, and 48.0-kD intermediates.

55.0, and 53.5 kD) that reach peak levels only upon 4 hincubation, and are degraded slowly so that they persist after12 h with the purified enzyme. Panel D shows the time courseof appearance of three intermediates (59.0, 50.0, and 48.0kD). The temporal profile of the 59.0-kD intermediate doesnot correspond to those of the groups depicted in panels Band C. This intermediate appears within a few minutes ofincubation, increases gradually to reach a plateau by 4 h, anddecreases slightly upon 12 h of incubation. By 24 h, however,the 59.0-kD intermediate is no longer evident in the incuba-tion mixture. The other two intermediates shown in panel D(50.0 and 48.0 kD) do not appear until after 2 h of incubation,and levels are still rising at 12 h of incubation. In fact, levelsof these intermediates after 24 h of incubation of the enzymewith 3-conglycinin are found to be even higher than at 12 h,suggesting that protease Cl does not degrade the 50.0 and48.0 kD intermediates, or does so with considerable difficulty.

Physiological Significance

Protease Cl was purified from the cotyledons of day 12plants, in which almost all ofthe storage proteins are digested.Does this enzyme play a role in the mobilization of storageprotein in vivo? The observation that this enzyme only con-stitutes 8 to 9% of the j-conglycinin-degrading activity shouldnot be of concern, because Figure 1 shows that its role in vivo

has terminated by day 12. The proportion of enzyme left ismore a matter of stability in comparison with the other j-conglycinin-degrading proteases. It is important, however,that the intermediates observed in the seedling cotyledons begenerated by the enzyme we have purified. In Table I, theintermediates generated upon the incubation off-conglycininwith purified protease Cl are compared with the intermediatesgenerated in vivo in the cotyledons, and with those producedin vitro by extracts from cotyledons harvested from seedlingsat different days of early growth. The table shows that manyof the same intermediates resulting from the degradation ofthe a' and a subunits of 3-conglycinin by protease C 1, suchas the 58.0-, 55.0-, 53.5-, 50.0-, and 48.0-kD intermediates,are also observed in vivo in the living plant. This observationsupports the idea that protease Cl plays an important rolein vivo.The absence of the larger proteolytic intermediates (74.0,

70.0, 65.0, 63.0, 61.0, and 59.0 kD) in vivo would indicatethat these proteolytic intermediates have a very transientexistence in the plant. We could only observe them in vitrowhen the proteolytic reactions were slowed down, using lessenzyme and performing the assays with short incubationtimes, as was done in the studies of the day 6 and day 12extracts and of the purified protease C 1. The fact that two ofthe major in vivo intermediates (51.8 and 45.0 kD) were notobserved in the in vitro degradation with purified protease Clsuggests that there are other proteolytic enzymes present invivo that also degrade f-conglycinin. Thus, although proteaseCl plays a significant role in f-conglycinin proteolysis, it isnot the only enzyme involved in the complex process ofmobilization of this storage protein.

DISCUSSION

Our results show that protease C1, the enzyme that wepurified from extracts of day 12 cotyledons, catalyzes theproteolytic degradation of the a' and a subunits of f-congly-cinin. Through this cleavage, a succession of intermediates ofmolecular mass 74.0-, 70.0-, 65.0-, 61.0-, 59.0-, 58.0-, 55.0-,53.5-, 50.0-, and 48.0-kD are generated. The sizes of most ofthese intermediates correspond to those observed upon incu-bation of f-conglycinin with extracts from the cotyledons ofday 1, day 6, and day 12 seedlings. Moreover, these includesome of the same intermediates observed in the cotyledonduring the first 3 d after imbibition. This correspondenceindicates that protease Cl, although purified here from day12 cotyledons, could be the same enzyme that plays the roleof initiating the mobilization of f-conglycinin during earlygrowth of the seedling. The continued presence of proteaseCl up to day 12 may be a consequence of the stability of theenzyme and/or of continued synthesis.From the temporal patterns of intermediates produced by

the proteolysis of d-conglycinin by protease C 1, a scheme forthis proteolysis may be hypothesized. The 74.0- and 65.0-kDintermediates are the first to appear, just as the a' and asubunits disappear. Thus, it is reasonable to conclude that theinitial proteolysis ofthe a' subunit yields the 74.0-kD product,which is 2 kD smaller than the original subunit. Similarly,one can hypothesize that the initial cleavage of the a subunit

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generates the 65.0-kD form, which is 1 kD smaller than thenative a subunit.With further incubation, the levels of the 74.0- and 65.0-

kD intermediates decrease, and the levels of the 55.0- and53.5-kD forms increase. It is unlikely that these new smallerforms are produced directly from the native subunits, as thea' and a subunits have totally disappeared before the 55.0-and 53.5-kD forms appear. After 4 h, the levels of the 55.0-and 53.5-kD intermediates decline, and the 50.0- and 48.0-kD forms appear. The levels of 50.0- and 48.0-kD forms are

found to increase up to 24 h of incubation. These same formsare also observed to be the main j3-conglycinin degradationintermediates in vivo, suggesting that they are the end productsof the limited proteolytic cleavage of the a' and a subunitsby protease C1. Further proteolysis is presumably catalyzedby other enzymes.

In summary, we propose the main pathways for p-congly-cinin degradation by protease C l to be:

a' subunit: 76.0 kD 74.0 kD 55.0 kD 50.0 kD

a subunit: 66.0 kD 65.0 kD 53.5 kD 48.0 kD

Other minor intermediates (70.0, 63.0, 61.0, 59.0, and 58.0kD) may be transient intermediates, or may be intermediatesarising from minor degradative pathways. Two prominentintermediates observed in vivo that are not generated byprotease Cl, 45.0 and 51.8 kD, presumably arise from suchalternative pathways. In fact, peaks 2 and 3 from the CM-Trisacryl column (Fig. 4) catalyzed the degradation of the a'and a subunits to the 45.0-kD intermediate. We have foundthe production of the 51.8-kD intermediate only upon incu-bation of fi-conglycinin with day 1 cotyledon extract.

Protease Cl exhibits an acidic pH optimum of 3.5 to 4.0,similar to other proteases shown to initiate the proteolysis ofstorage protein in Vigna radiata (1), Vicia sativa (4), Phas-eolus vulgaris (3), and Vigna mungo (14), and the glycininand the KSTI-degrading enzymes in soybean (29). All oftheseother proteases are cysteine proteases, however, and may beconsidered to be "protease A"-type enzymes (20). In contrast,protease Cl is a serine protease. Until now, the only otherplant serine protease that has been shown to play a physiolog-ically relevant role in seed protein mobilization is proteinaseF, which initiates the degradation of the trypsin inhibitor inVigna radiata (28). Proteinase F, like the soybean proteaseC 1, has an acidic pH optimum, making this class ofplant serine proteases quite distinct from the more familiaranimal serine proteases that typically function at neutral toalkaline pH.

It is not surprising that protease Cl catalyzes the degrada-tion of both the a' and a subunits of j3-conglycinin, consid-ering that there is a 93% amino acid sequence identity be-tween these two subunits (18). The ,B subunit, in contrast, ismissing a segment of 179 amino acid residues at the aminoterminus compared with the a' and a subunits (25). Asidefrom this missing segment, however, there is still about 75%sequence identity between the A subunit and the a' subunit(25). The fact that protease Cl does not act toward the asubunit suggests that the protease C cleavage sites on the a'and a subunits are outside the region of sequence homologyshared by the three subunits, and thus, by inference, in the

amino terminal region of the a' and a subunits. Because theinitial cleavage by protease Cl occurs with the loss of 1 to 2kD in molecular mass, we hypothesize that this first cleavageoccurs between residues 10 to 20 in the amino termini of thea' and a subunits. Subsequent cleavages by protease Clproduce limiting products of 50.0 and 48.0 kD. We hypoth-esize that these limiting products correspond to the region ofhomology shared by the three subunits, extending up to thecarboxyl termini. This carboxyl terminal segment is somewhatlonger in the a' subunit than in the a subunit, and wouldexplain the size difference between the 50.0- and 48.0-kDlimit products.These observations suggest that the action of protease Cl

on #-conglycinin mobilizes up to one-third of the a' and asubunit amino acid residues. The observation of the proteo-lytic products of protease Cl action as early as day 1 showsthat this pathway is one of the earliest generators of aminoacids for the germinating seed. Limitation of this action ofprotease C1, such that the 50.0- and 48.0-kD intermediatesas well as the f subunit cannot be degraded, ensures that theamino acids stored in 3-conglycinin are not all released inone early burst, but rather are made available over a longerperiod of time. Thus, the soybean seedling is ensured botha quick, early supply of amino acids as well as a supply last-ing until such time as the plant becomes a photosyntheticautotroph.

LITERATURE CITED

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28. Wilson KA, Tan-Wilson AL (1987) Characterization of the pro-teinase that initiates the degradation of the trypsin inhibitor ingerminating mung beans (Vigna radiata). Plant Physiol 84:93-98

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Characterization of the Major Protease Involved the ... Chromatography ... The gel, shown in Figure 1, was analyzed for the degradationof,B-conglycinin subunitsandfortheappearance