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Plant Physiol. (1983) 72,
345-3500032-0889/83/72/0345/06/$00.50/0
Characterization of Soybean Endopeptidase Activity
UsingExogenous and Endogenous Substrates'
Received for publication October 21, 1982 and in revised form
January 19, 1983
HEATHER M. BOND AND DIANNA J. BOWLESDepartment of Biochemistry,
University of Leeds, Leeds, LS2 9JT United Kingdom
ABSTRACT
Endopeptidase activity in mature soybean seeds (Glycine max),
has beenmeasured using an exogenous substrate, 1125lliodoinsulin B
chain. On thebasis of pH optimum and the use of specific proteinase
inhibitors, twodistinct endopeptidase activities can be identified
in both the embryonicaxis and the cotyledons. One activity is
characteristic of a neutral/alkalinemetalloendopeptidase(s) and the
other of an acidic carboxylendopepti-dase(s). Neither activity is
membrane-bound. The metalloendopeptidase(s),most probably working
with neutral expopeptidases also present in thetissues, is capable
of degrading certain subunits of the storage proteins.The a subunit
of conglycinin and additional seed polypeptides remainresistant to
degradation. The carboxylpeptidase activity displayed a differ-ent
specificity towards endogenous substrates; in particular, an
acid-solublepolypeptide of apparent molecular weight 30,000
appeared to be theprincipal substrate for limited proteolytic
degradation by the proteinase(s).Soybean agglutinin remained
resistant to degradation by either class ofendopeptidases.
Storage reserves in soybeans are composed of specific
polypep-tides localized in the cotyledons and axes of the mature
seeds.Much recent work has focused on the characterization of the
seedstorage proteins, including the regulation of their synthesis
duringseed maturation and the processes that lead to their
eventualdeposition in the protein bodies (14, 16, 19-21). In
contrast, verylittle information is available concerning the
mechanism of soy-bean storage protein degradation and the events
that regulategermination and mobilization of reserves during the
early stagesof seedling growth (9).As a first step into this
complexity, we have now characterized
the endopeptidase activity that exists in cotyledons and axes
ofmature soybean seeds. We present evidence to indicate there
aretwo distinct classes of endopeptidases, one with an acidic
pHoptimum and characteristics of carboxylpeptidase activity,
theother with a neutral/alkaline pH optimum and typical of
metal-loendopeptidase activity. The neutral metalloendopeptidase
activ-ity, working most probably in conjunction with exopeptidases
alsopresent in the tissue, is able to degrade the storage
polypeptides invitro with the exception of the ,8-subunit of
conglycinin and thebasic subunits of glycinin.
MATERIALS AND METHODS
Soybean seeds (Glycine max var. Williams) were provided bythe US
Intsoy program 1979, with a germination efficiency of
'This work was financially supported by an Science Research
CouncilGrant A 97783 and B 73088 to Dr Dianna J. Bowles.
100%. I251-labeled B chain of insulin was a gift from Dr. A.
J.Kenny. All chemicals, unless otherwise stated, were from
Sigma.Soybean agglutinin was prepared from affinity
chromatographyas described in Allen and Neuberger (2).
Preparation of Soybean Seed Extracts. For determination
ofendopeptidase activity in dry seeds, cotyledons were dissected
andground first to a fine powder in a coffee grinder.
Otherwise,soybeans were soaked for 4 h in H20 at room temperature
anddissected into cotyledons and axes. The material from either
dryor imbibed seeds was then homogenized in a pestle and mortar
atthe fresh weight: buffer ratio of 1 g:10 ml. The buffers used
werePBS,2 100 mm Hepes/NaOH pH 8.0, or 100 mm NaCH3COO/CH3COOH, pH
4.0. The homogenates were sonicated on ice (4x 10 s, using a
microprobe fitted to a Dawe Sonicator), centrifugedfor 5 min on a
bench centrifuge to provide a debris-free superna-tant which was
used to assay for protein and endopeptidaseactivity.
Endopeptidase Assay. [125JIlodoinsulin B chain is hydrolyzed
torelease TCA-soluble radioactive peptides. The assay was
carriedout essentially as described in Barrett (4) and George and
Kenny(13). For determination of pH optima, the following buffers
wereused: pH 2.3, 3.0, and 4.0, 100 mm NaCH3COO/CH3COOH; pH5 and 6,
100 mm Mes/HCl; pH 7 and 8, 100 mm Hepes/NaOH;pH 9 and 10, 100 mm
Tris/HCl. The assay contained 200 pl ofappropriate buffer, 50 ,l of
extract, and H20 and/or potentialinhibitors to a final volume of
275 1d. The reaction was startedwith 25 ,ul 1251-labeled B chain at
5.7 nmol/ml.
After the incubations at temperature and time specified,
thereaction was terminated by the addition of 500 ,ul TCA (25%
w/vin H20) and 250 id casein (2% w/v in H20). After
centrifugationat l0,000g for 3 min (Eppendorf Microfuge), 500-Al
aliquots wereremoved and counted in a y-counter and the per cent
degradationof '25I-B chain was measured. After obtaining total
radioactivitypresent and TCA-soluble radioactivity, in the absence
ofenzyme/extract, nmol 125I-B chain degraded was calculated as
described inGeorge and Kenny (13).When measuring the effect of
potential inhibitors on endopep-
tidase activity, the extract, appropriate buffer, and inhibitor
werepreincubated for 2 h at 4°C prior to addition of 1251I-B chain
andstart of assay.
Aminopeptidase Assay. Activity was measured
fluorometricallyusing AMC derivatives of amino acids as substrates
in an assayessentially the same as that used for 2-naphthylamide
derivatives(4). The fluorimeter cell contained 1.9 ml of buffer
substratemixture equilibrated at 37°C. Buffer substrates used were:
Ala-AMC, 0.2 mm in 100 mm Tris/HCl, pH 7.0; Gly-Pro-AMC, 0.2
2Abbreviations: PBS, phosphate-buffered saline (10 mm
KH2PO4/K2HPO4, 145 mm NaCl, pH 7.4), pCMB, p-chloromercuribenzoic
acid;PMSF, phenylmethylsulfonyl fluoride; AMC,
7-amino-4-methylcoumarin;STI, soybean trypsin inhibitor, SBA,
soybean agglutinin; PAGE, poly-acrylamide gel electrophoresis.
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Plant Physiol. Vol. 72, 1983
mm in 100 mm Tris/HCl, pH 8.0; Glu-AMC, 0.2 mm Tris/HCl,pH 7.0,
containing 1.25 mm CaCl2. The reaction was started byaddition of
100 I1 of soybean extract (made in Hepes/NaOH, pH8.0, at a protein
concentration of 10 mg/ml). The increase inemission was measured
over 3 min in the cell maintained at 37°C(excitation, 370 nm, slit
0.5; emission, 442, slit 2).
General Methods. Protein was determined using the Coomassiedye
method (26). For analysis of polypeptides using polyacryl-amide
electrophoresis in the presence of SDS, samples wereprecipitated
using 12.5% (w/v) TCA (final concentration) andresolubilized in
sample buffer and processed using 10 to 15% (w/v) gradient gels as
described in Bowles and Marcus (7). Ultracen-trifugation of the
extracts was performed using an SW 50.1 rotortype in a Kontron
ultracentrifuge at 4°C for 30 min at 100,000g.Hemagglutination
assays using trypsin-treated rabbit erythrocytes,were performed as
described in Bowles and Marcus (7).
RESULTS
Characterization of Endopeptidase Activity Using an
ExogenousSubstrate. The initial experiments were based on the
ability of atissue extract to hydrolyze an exogenous 125I-labeled
substrate ina standard endopeptidase assay. Using this approach,
severalparameters of endopeptidase activity in soybeans were
examined.Figure 1 shows the pH optima of activity in extracts
preparedfrom axes (A) and cotyledons (B) of 4-h-imbibed seeds. For
bothtissues, two distinct optima exist, one at pH 4.0 and the other
atpH 8.0. At either pH, the specific activity is higher in the axis
thanin the cotyledon. A time course for degradation of the
exogenoussubstrate is shown in Figure 2. Hydrolysis of 1251-B chain
insulinis linear up to 50 min at 37°C.A range of additives were
included in the standard assay to gain
an insight into the class of endopeptidase activity present in
thetissues. Table I shows the results of the additions to tissue
extractsprepared at pH 4.0 and 8.0. Cotyledon and axis extracts
respondin a similar manner to all the additives. However, a major
distinc-tion exists in the response to the activity measured at pH
4.0compared with that measured at pH 8.0. Endopeptidase activityat
pH 8.0 is inhibited by a range of chelators and metal ionsincluding
1,10-phenanthroline, EDTA, Cu2+, and Zn2+. This pat-tern of
inhibition is typical of that shown by metalloendopepti-dases which
generally exhibit a pH optimum in the range of 7 to9 (4). The
inhibition by pCMB and DTT also indicates theinvolvement of thiol
groups in the activity. PMSF interacts bothwith thiol groups and
serine proteases (4). Since other serineprotease inhibitors were
without effect on the activity at pH 8.0,PMSF inhibition may also
be related to the involvement of thiolgroups. Certain metal ions
including Ca2" and Mn2+ stimulatedactivity. However, since the
tissue extracts most probably con-tained endogenous metal ions and
chelators, the specific interac-tion between the enzyme and metal
ions must await the purifica-tion and detailed analysis of the
enzyme(s). In contrast to thepattern of inhibition at pH 8.0, the
activity measured at pH 4.0was unaffected by metal ions and
chelators but was abolished bypepstatin A. The pH optimum and
specific inhibition are typicalof carboxylpeptidases that generally
have a pH range of 2 to 5.Aprotinin was also able to inhibit the
activity measured at pH 4.0,although it is generally assumed to be
specific for trypsin andserine proteases. However, other serine
proteinase inhibitors in-cluding benzamidine were without effect on
the activity. Neitherthe presence of excess SBA nor its removal by
passage of theextracts through an affinity matrix affected
endopeptidase activityat either pH optimum. It is therefore
unlikely that the lectin isdirectly involved through its
carbohydrate-binding site with theregulation of endopeptidase
activity.
Effect of Endopeptidase Activity on Endogenous Substrates. Itwas
of interest to determine the effect of endopeptidase activityon
soybean proteins. Extracts prepared from cotyledons and axes
.' 0
150
.1
S so
Co
50-
c 44
IC.15
R la
*e5
gopH
B
I I I I I I I I2 3 4 5 6 7 8 9 10
pHFIG. 1. pH optima of endopeptidase activity in extracts of
soybean
axes and cotyledons. Extracts were prepared from the embryonic
axis (A)and the cotyledon tissue (B) in PBS as in "Materials and
Methods" andassayed at 37°C for 15 min, using ['"Iliodoinsulin B
chain as a substrateover the pH range 2.3 to 10.0.
were incubated for 24 h at 30°C at either pH 4.0 or 8.0 in
thepresence/absence of inhibitors of endopeptidase activity.
Usingthe exogenous "2I-labeled substrate, it was shown that both
classesof endopeptidase activity were stable for 24 h at 30°C and
theeffect of the inhibitors was maintained throughout the
incubation(results not shown). After the incubation period, samples
wereanalyzed by polyacrylamide gel electrophoresis in the presence
ofSDS and compared with the polypeptide profile of the
tissueextracts at time 0. The results for cotyledon extracts are
shown inFigure 3 and those for embryonic axis extracts are in
Figure 4.The extract of the cotyledons prepared at pH 8.0 and time
0 (Fig.3, lane A) contains the subunits of the major storage
polypeptides:glycinin (llS: A-4, acidic, and basic subunits) and
conglycinin(7S: a'-, a-, and fl-subunits). Extraction of the
cotyledons at pH
I I I
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SOYBEAN ENDOPEPTIDASES
A
t 6
'RIO1
30 60 90 120Minutes
B
5
.a 4 ~ 03
30 60 90 120Minutes
FIG. 2. Time course of endopeptidase activity in extracts of
soybeanaxes and cotyledons. Extracts were prepared from the axis
(A), andcotyledon (B) at pH 8.0 or 4.0 and as described in
"Materials and Methods"were assayed for endopeptidase activity
using ['25Iliodoinsulin B chain asa substrate for times up to 2 h
at 370C.
4.0, time 0, (Fig. 3, lane E) causes the selective precipitation
of thestorage proteins and much other material (-75% of the
totalprotein) resulting in an enrichment in certain acid-soluble
poly-peptides including urease.
Incubation of the cotyledon extract at pH 8.0 for 24 h in
theabsence of endopeptidase inhibitors causes a massive alteration
tothe polypeptide profile (Fig. 3, lane B). The A-4 and
acidicglycinin subunits and the a'- and a-subunits of conglycinin
areabsent. Many new low mol wt bands appear, including
prominent
Table I. Effects ofAdditives on Endopeptidase Activity in
ExtractsPreparedfrom Soybean Cotyledons and Axes
Extracts prepared at pH 4.0 or 8.0 were preincubated at 4°C for
2 h inthe presence of different additives, before inclusion of
['25Iiodoinsulin Bchain. Endopeptidase activity was then measured
over a 30-min period asdescribed in "Materials and Methods." TIU,
trypsin-inhibiting units.
Cotyledon Embryonic Axis
Additive Concn. of Ho saInhibitor pH of assay
4.0 8.0 4.0 8.0
% activity remainingNo addition 100 100 100 100CuCl2 2 mM 102.3
0 69.1 0ZnCl2 2 mm 93.9 68.6 77.4 38.0MnCl2 2 mM 127.0 183.0 81.3
115.0MgCl2 2 mM 96.5 103.1 65.4 116.0CaCl2 2 mm 110.6 176.0 60.9
92.9EDTA 5 mM 100.7 10.7 79.7 62.4DTT 2 mM 100.5 12.5 79.4
45.71,10-Phenanthroline 5 mm 45.2 0 70.7 0pCMB I mM 87.0 0 63.5
0lodoacetimide 2 mm 106.2 95.5 69.8 108.0PMSF I mm 49.4 52.0 39.3
84.9STI 250 yg/ml 109.4 107.8 96.2 94.0Benzamidine I mm 100.3 99.6
113.1 102.0Aprotinin 2.83 TIU/ml 10.4 63.9 0 103.9Pepstatin A 5
,tg/ml 2.5 109.0 0 123.7SBA 250 tig/ml 109.4 107.8 96.2 94.2Minus
SBA 89.8 109.9 100.6 91.3
ones at apparent mol wt 48,000, 33,000, 29,000 and 26,000.
Theresults suggest that the fl-subunit of conglycinin is unaffected
bythe incubation as are the basic subunits of glycinin. However,
thepossibility cannot be ruled out that degradation products of
iden-tical apparent mol wt to these subunits are formed and cannot
bedistinguished on a one-dimensional gradient gel. Inclusion
ofeither 1 mM pCMB (Fig. 3, lane C) or 5 mm
1,10-phenanthroline(Fig. 3, lane D) completely prevents degradation
and the polypep-tide profiles of the incubations with either of
these inhibitorspresent remain unchanged from that at time 0.The
effect of a 24-h incubation of the cotyledon extract at pH
4.0, in the absence ofendopeptidase inhibitors, is far less
dramatic(Fig. 3, lane F). The apparent mol wt of urease may be
slightlyreduced, but certain other unidentified polypeptides
disappearcompletely, including a prominent band at apparent mol wt
30,000and several minor ones. A new lower apparent mol wt
bandappears at 23,400. In the presence of either 5 jg/ml pepstatin
or10%o (v/v) aprotinin, the polypeptide at apparent mol wt 30,000
ismaintained and the main degradation product is absent. The
effectof the two inhibitors seems to be similar: neither is able to
preventdegradation of several minor polypeptides in the extract.
Thepolypeptide in Figure 3, lane G, of apparent mol wt 6,500
isaprotinin.As shown in Figure 3, the storage proteins precipitated
in 100
mm acetate buffer at pH 4.0 and were generally removed
bycentrifugation before assaying the effect of the acidic
carboxyl-peptidase activity. In an attempt to determine whether the
acidicendopeptidase could also degrade the storage proteins, a
cotyledonextract prepared under neutral conditions was reduced to
pH 4.0and the suspension was incubated with shaking at 30°C for 24
h.No significant alteration to the polypeptide profile occurred
(re-sults not shown).The data therefore suggest that degradation of
the storage
proteins of the cotyledons can occur in vitro at pH 8.0 and
beprevented by inhibitors of the neutral metalloendopeptidase
activ-
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Plant Physiol. Vol. 72, 1983
-40
FiG. 3. SDS-PAGE of soybean cotyledon extracts incubated at 300C
for 24 h in the presence/absence of endopeptidase inhibitors.
Cotyledonextracts were prepared at pH 8.0 (A-D) and 4.0 (E-H). Time
0, A and E; 24-h incubation in the absence of inhibitors, B and F;
24 h in the presence ofinhibitors, C, D, G, and H. C, 1 mM pCMB; D,
5 mM 1,10-phenanthroline; G, 10% (v/v) aprotinin; and H, 5 pg/ml
pepstatin A.
ity. However, it was possible that degradation of the
polypeptidesalso involved the action ofexopeptidases which
exhibited a similarpH optimum and characteristics of inhibition. As
a preliminarystep into testing this possibility, several synthetic
AMC-aminoacid derivatives were used to assay for aminopeptidases
and theirsusceptibility to pCMB and 1, l0-phenanthroline. The
results areshown in Table II and indicate that soybean cotyledon
extracts docontain substantial levels of aminopeptidases which are
inhibitedby 1, l0-phenanthroline and to a lesser extent by
pCMB.
Figure 4 shows the effect of endopeptidase activity on
embry-onic axis polypeptides. Extraction of the axis at pH 8.0
(time 0,Fig. 4, lane A) revealed a markedly different profile to
that of thecotyledon. Although bands corresponding in apparent size
to themajor storage proteins, glycinin and conglycinin, were
present,their amounts in the axis were much lower than in the
cotyledons.As before, extraction of the tissue at pH 4.0 (time 0,
Fig. 4, laneE) caused precipitation of many polypeptides.The effect
of a 24-h incubation of the axis extracts in the
absence of proteinase inhibitors was less dramatic than for
thecotyledons but the overall pattern of change remained similar.
AtpH 8.0, a- and a-subunits ofconglycinin and glycinin
disappeared(Fig. 4, lane B) and were retained in the presence of
proteinaseinhibitors (Fig. 4, lanes C and D). At pH 4.0, the major
substratefor proteolytic activity might again be a polypeptide at
apparentmol wt 30,000, inasmuch as the most notable change in the
extractwas the appearance ofa probable degradation product at
apparentmol wt 23,400 (Fig. 4, lane F) and its absence if the
incubationcontained inhibitors (Fig. 4, lanes G and H).SDS-PAGE
analysis provided an overall insight into the sus-
ceptibility of abundant proteins to degradation by
endogenousproteinases. However, SBA which constitutes only
-
SOYBEAN ENDOPEPTIDASES
A B C D E F G Hw _ WIW
Table III. The Effect of Ultracentrifugation on the Solubility
ofEndopeptidase Activities in Extracts Preparedfrom Soybean
CotyledonsCotyledons were homogenized at pH 4.0 or 8.0 and
centrifuged at low
speed to obtain a debris-free extract as described in "Materials
andMethods." This extract was then further centrifuged at l00,000g
for 30min at 4VC. Endopeptidase activities before and after
ultracentrifugationwere assayed using I'25lliodoinsulin B chain as
a substrate.
Precentrifugation Exract PostcentrifugationCotyledon
Supernatant
pH of Preparation B Chain o B ChainProtein Hydrolyzed Protei
Hydrolyzed
mglml nmoi/min.mg mg/ml nmol/min mgprotein protein8.0 7.6 0.126
6.2 0.1544.0 2.4 0.562 1.6 0.5 12
i
FIG. 4. SDS-PAGE of soybean axes extracts incubated at 30'C for
24hours in the presence/absence of endopeptidase inhibitors. Axes
extractswere prepared at pH 8.0 (A-D) and 4.0 (E-H). Time 0, A and
E; 24-hincubation in the absence of inhibitors, B and F; 24 h in
the presence ofinhibitors, C, D, G, and H. C, I mm pCMB; D, 5 mM
1,10-phenanthroline;G, 10%1b (v/v) aprotinin; and H, 5 jig/ml
pepstatin A.
Table II. Aminopeptidase Activities in Extracts Preparedfrom
SoybeanCotyledons
Exogenous 7-amino-4-methylcoumarin amino acid derivatives
wereused as substrates and assayed fluorimetrically using a soluble
extractprepared from 4-h-imbibed seeds homogenized in 0.1 M Hepes,
pH 8.0, asdescribed in "Materials and Methods."
Substrate Inhibitor AMC Hydrolyzed
mM nmol/min .mgproteinAla-AMC 6.28
l,1O-Phenanthroline (5) 0.40pCMB (1) 3.00
Gly-Pro-AMC 0.2531, 10-Phenanthroline (5) 0.16pCMB (1) 0.216
a-Glu-AMC 0.0841, 10-Phenanthroline (5) 0.028pCMB (1) 0.022
change in TCA solubility of iodinated residues (13). The
natureof the substrate is such that it is specific for endo- rather
thanexopeptidase activity and has been used widely to
characterizeproteinases of mammalian systems (4). Endopeptidases
are clas-sified into distinct groups on the basis of their pH
optima and,more definitively, on the effect of specific substances
to inhibittheir activity (4). Using these criteria, cotyledons and
axes ofsoybean contain neutral metalloendopeptidase activity and
acidiccarboxylpeptidase activity.
Previously, when Na-benzoyl-DL-arginine-p-nitronilide was
used as exogenous substrate, only a single neutral (pH
8.2-8.5)peptidase activity was found (8, 15). In earlier studies
(19, 22), theuse of casein as substrate indicated a heterogeneous
population ofpeptidase activities, all with pH optima in the range
5.0 to 6.0 (17,27, 31). Given the limitations of these substrates
and assays tospecifically detect endopeptidase activity and the
absence of anydetailed inhibitor data in previous investigations,
it is unclear towhat extent the present data correspond with
earlier findings.A major weakness in using any exogenous substrate
in vitro is
that it might not reflect proteolytic activity of relevance in
vivo.Therefore, as a preliminary to enzyme purification, the
inhibitorsof the metallo- and carboxylendopeptidases were used to
studythe effect of the proteinases on soybean proteins. The
resultsindicate that the two proteolytic activities are each able
to degradespecific seed proteins in vitro. Under neutral pH
conditions, inwhich the major storage proteins are soluble and the
metalloen-dopeptidase(s) is active, certain storage polypeptides
are com-pletely degraded whereas others remain unaltered. Thus, the
a'-,a-, A4, and acidic subunits of conglycinin and glycinin,
respec-tively, are degraded, whereas the ,B-subunit of conglycinin
and thebasic subunits of glycinin remain intact. We suggest the
differentsubunits of the storage reserves may possess distinct
physiologicalfunctions in vivo. Interestingly, the ,B-subunit of
conglycinin ap-pears later in seed maturation (22 d after
flowering) than the a'-and a-subunits (15-17 d after flowering)
(14). Although formed atseparate stages of embryogenesis, the
different subunits still asso-ciate to form seven naturally
occurring isomeric multimers in themature seed (27). Multimer
formation may, however, only reflectan efficiency in packaging the
reserves into protein bodies forstorage until germination. Evidence
suggests that the carboxylpep-tidase may not be involved in the
degradation of storage reserves,and, at least in the early stages
of germination, some mobilizationto amino acids could occur through
only the combined action ofthe metalloendopeptidase(s) and
exopeptidases. The principal sub-strate for the
carboxylpeptidases(s) is a prominent unidentifiedpolypeptide of mol
wt 30,000 which occurs in both the axis andcotyledons. Proteolysis
of the polypeptide appears to be limited inthat its disappearance
coincides with the appearance of a newband of lower mol wt which
could represent one of the fragmentsof the original substrate.
However, tryptic maps would be neces-sary to confirm their
relatedness. The carbohydrate-binding activ-ity of SBA is not
affected by endopeptidase activity at either pH8.0 or 4.0. This
suggests the lectin is not used as an immediatesource of nitrogen
for the growing axis and confirms the proba-bility that lectins
cannot be classified strictly as storage reserves inthe seed (3, 6,
11, 31).Our results indicate that the specific activity of the
proteinases
in extracts prepared from axes and measured using iodoinsulin
Bchain is greater than in cotyledon extracts. This seems
surprising
349
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since the cotyledons are envisaged as the main autolytic
centersduring germination and early stages of seedling growth.
Onepossible explanation could be competition from endogenous
sub-strates which might be at a higher level in the cotyledons than
inthe axes. A comparison of activities using iodoinsulin B
chainafter a 24-h incubation in vitro, during which depletion of
endog-enous substrates had occurred, still revealed the axis was
higherthan the cotyledon. However, it is probable that neither
class ofendopeptidase is functioning optimally in the tissue
extracts dueto the presence/absence ofinhibitors/cofactors, etc.
and, therefore,no idea of the full proteolytic potential of the
tissues will beobtained until the enzymes have been purified and
antisera areavailable.The use of tissue extracts can only provide
an indication of the
probable events that occur in vivo. When polypeptide profiles
ofcotyledon and axis extracts prepared from 24-h-imbibed seedswere
investigated by SDS-PAGE, essentially no alteration inprofiles from
time 0 had occurred (results not shown). This mayreflect an
acceleration in degradation brought about by the con-ditions of the
in vitro incubation. Alternatively, it is possible thatneither
endopeptidase activity present in mature soybeans is re-sponsible
for the physiological mobilization of reserves duringgermination.
For example, in the two well characterized systemsof mung bean and
castor bean, the proteinases responsible formobilization of storage
reserves are only synthesized 2 to 3 d afterseed germination (1, 5,
10, 23, 24, 28, 30). However, our dataindicate that proteinases
which have been synthesized duringsoybean maturation have at least
the potential to degrade specificstorage polypeptides. As yet, we
have no evidence whether theprimary function ofthese proteinases is
in storage protein synthesisand processing (12) or reserve
mobilization.
LITERATURE CITED
1. ALPI A, H BEEVERS 1981 Protelnases and enzyme stability in
crude extracts ofcaster bean endosperm. Plant Physiol 67:
499-502
2. ALLEN AK, A NEUBERGER 1975 A simple method for the
preparation of anaffinity absorbent for soybean agglutinin using
galactosamine and CH-Seph-arose. FEBS Lett 50: 362-363
3. BARONDES SH 1981 Lectins: their multiple endogenous cellular
functions. AnnuRev Biochem 50: 207-231
4. BARRr AJ (ed) 1977 Proteinases in mammalian cells and
tissues. In ResearchMonographs in Cell and Tissue Physiology, Vol
2. Elsevier/North-HollandBiomedical Press, pp 1-52 (Barrett) and pp
436-440 (AJ Kenny)
5. BAUMGARTNER B, MJ CHRISPELS 1977 Purification and
characterization ofvicilin peptidohydrolase the major endopeptidase
in the cotyledons of mungbean seedlings. Eur J Biochem 77:
223-233
6. BowLEs DJ 1982 The enigma of seed lectins. Recherche 13:
784-785
7. BowLEs DJ, S MARCUS 1981 Characterization of receptors for
the endogenouslectins of soybean and jackbean seeds. FEBS Lett 129:
135-138
8. CATSIMPOOLAS N, SK FUNK, J WANG, J KENNY 1972 Isoelectric
fractionationand some properties of a proteases from soybean seeds.
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