Insect Biochem. Vol. 19, No. 3, pp. 221-231, 1989 0020-1790/89 $3.00+ 0.

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Printed in Great Britain. All rights reserved Copyright © 1989 Pergamon Press pie

PARTIAL PURIFICATION A N D CHARACTERIZATION OF

THE MAJOR MIDGUT PROTEASES OF GRASS GRUB LARVAE
(COSTEL YTRA ZEALANDICA, COLEOPTERA: SCARABAEIDAE)
J. T. CHRISTELLER 1, B. D. SHAW1, S. E. GARDINER l and J. DYMOCK 2
'Plant Physiology Division, DSIR, Palmerston North, New Zealand and 2Entomology Division, DSIR,
Palmerston North, New Zealand

(Received 24 June 1988; revised and accepted 5 October 1988)

Abstract--The major proteases of the grass grub (Costelytra zealandica) larval midgut have been
identified, partially purified and characterized. Identification was made initially on the basis of hydrolysis
of synthetic substrates (blocked and partially blocked esters and amides of specific amino acids), thus
classifying the activities into different classes of endo- and exopeptidases. A range of inhibitors specific
to different classes of proteases were used to confirm the presence of trypsin, chymotrypsin, elastase,
leucine aminopeptidase and carboxypeptidases A and B and to establish the absence of thiol- and
metallo-endopeptidases. The dominant endopeptidase in the midgut is trypsin, which is present in four
forms, distinguishable by net charge, but indistinguishable either in terms of Michaelis-Menten param-
eters (Km and kcat) or in molecular weight (23,000). The pH optimum lies between pH 9-10. Leucine
aminopeptidase has a molecular weight of 91,000 and a pH optimum at pH 8.0. Carboxypeptidase A has
a molecular weight of 43,000 and a pH optimum at pH 8.5. All enzymes retained substantial activity at
pH 7.0-7.1, the pH of the midgut lumen, where the bulk of the activity was located. Protease levels in
the hindgut (or fermentation sac) were 1-5% of those in the midgut. The range of enzymes appears
sufficient for complete breakdown of ingested protein.

Key Word Index: Costelytra zealandica, grass grub, gut, protease, Coleoptera, Scarabaeidae, leucine
aminopeptidase, carboxypeptidase, trypsin, chymotrypsin, elastase

INTRODUCTION grub and only limited information about its digestive

physiology, mostly in the area of carbohydrate di-
The white grub complex of coleopterous larvae of the gestive capabilities, has been sought (Bauchop and
sub family Melolonthinae are economically important Clarke, 1975). Currently, very little insecticide is
pests in many parts of the world (e.g. King, 1984; applied due to cost and alternative forms of control
Vittum, 1986; Wood, 1977). Costelytra zealandica are needed.
(White) is the only important endemic representative One possible alternative has been demonstrated by
of this complex in New Zealand. The larvae cause the efficacy of a proteinaceous proteinase inhibitor as
considerable damage to pastures, living in the soil a resistance factor against tobacco budworm larvae
and feeding on the roots of grasses and legumes. (Heliothis virescens, Lepidoptera) (Hilder et al., 1987)
Insecticides were used for many years to control grass in transgenic tobacco. The approach is supported by
several feeding studies of insects on artificial diets
Abbreviationa used: BAEE, N~t-benzoyl-L-arginine ethyl (Gatehouse and Boulter, 1983; Broadway and
ester; BApNA, Na-benzoyl-DL-arginine p-nitroanilide; Duffey, 1986; Shade et al., 1986; Wolfson and Mur-
BANA, N-benzoyl-DL-arginine p -naphthylamide; dock, 1987) or following herbivory where natural
BTEE, N-benzoyl-L-tyrosine ethyl ester; BTpNA, N- proteinase inhibitor levels are increased (Broadway et
benzoyl-L-tyrosine p-nitroanilide; HA, hippuryl- al., 1986).
L-arginine; HPLA, hippuryl-DL-phenylactic acid; Extracellular proteases, either free in the gut lumen
SApNA, N-suecinyl-ala-ala-ala p-nitroanilide; BSA, or bound to microvillar membranes, arc responsible
bovine serum albumin; PCPI, potato tuber carboxy-
peptidase inhibitor; CEOM, chicken eggwhite ovo- for degrading dietary protein in insects (Applebaum,
mucoid; SBTI, soybean trypsin inhibitor; LBTI, lima 1985; Santos et al., 1986). The characteristic classes
bean trypsin inhibitor; CPTI, cowpea trypsin inhibitor; of endopeptidases present seem matched to the pH of
SBBI, soybean Bowman-Birk inhibitor; PMSF, phenyl- the insect gut lumen. Serine proteinases are present
methylsulphonyl fluoride; TLCK, N~t-p-tosyl-L-lysine under neutral or mildly alkaline conditions and thiol
chloromethyl ketone; E-64, trans-epoxy succinyl- and aspartic proteinases under neutral to mildly
L-leucylamido-(4-guanidino)-butane; IAA, iodo- acidic gut contents (Houseman and Downe, 1982;
acetamide; DTT, dithiothreitol; NEM, N-ethyl- Kitch and Murdock, 1986; Murdock et ai., 1987;
maleimide; DTNB, 5,5'-dithiobis-(2-nitrobenzoic acid); Wolfson and Murdock, 1987). Within coleoptera, the
PIPES, pipcrazine-N,N'-bis-[2-ethanesulphonic acid];
MES, 2-[N-morpholino]ethanesulphonic acid; MOPS, subject of the current paper, digestive systems based
3-[N-morpholino]propanesulphonic acid; PVPP, poly- predominantly on serine proteinases or thiol pro-
vinylpolypyrrolidone; SDS--PAGE, sodium dodecylsol- teinases have been found (Baker, 1981; Kitch and
phate-polyacrylamide gel electrophoresis; DMF, N,N- Murdock, 1986; Murdock et ai., 1987).
dimethyl formamide; TCA, trichloroacetic acid. Before assessing the potential use of protease in-
LB. 191~^ 221
222 J.T. CHRISTELLERet al.
hibitors to provide resistance against grass g r u b in
pasture species, we investigated the properties o f the
m a j o r digestive proteases of grass g r u b larvae.
The enzyme n o m e n c l a t u r e o f Storey a n d W a g n e r
(1986) has been a d o p t e d t h r o u g h o u t .
MATERIALS AND M E T H O D S
Materials
The substrates BApNA, BTpNA, BANA, LpNA,
SApNA, BAEE, BTEE, HPLA and HA were obtained from
Sigma, as well as bovine trypsin, bovine chymotrypsin,
carbonic anhydrase, ovalbumin, BSA, cytochrome c, PCPI,
SBTI, CEOM, aprotinin, cystatin, elastin-orcein and the
molecular weight standards for SDS-PAGE. R. rubrum
ribulose bisophosphate carboxylase was purified from E.
coil containing the plasmid pRE41 (Terzaghi et al., 1986).
PMSF, TLCK, Antipain, E-64, Fast Black K salt and Blue
Dextran were obtained from Sigma. Other reagents were
of analytical or reagent grade, or obtained as gifts (see
Acknowledgements).
Preparation of crude extracts
Third instar larvae were obtained from the field from
April to June. The larvae were anaesthetized with COe and
the midgut sections removed at 4°C and stored at - 8 0 ° C
until required. No changes in extractable activity of major
proteases have been detected after 9 months storage.
Extracts were prepared by homogenization of guts in
50mM Tris-HC1, pH 8.0 (3-5 guts ml -~) at 4°C. The
supernatant from centrifugation at 10,000g, 10min was
used as the crude source of gut proteases for character-
ization and purification. No changes in gut protease activ-
ities occurred during storage of crude extracts at 4°C for 5
days. No decrease in trypsin activity in pooled DEAE-
purified fractions has been detected after storage in buffer
for 6 months at 4°C or in 25% glycerol at -20°C.
Purification
Typically 50-100 larval guts were homogenized in
10-20 ml 50 mM PIPES, pH 6.8 and centrifuged at 15,000g,
10 min before applying the supernatant to a small DEAE
column equilibrated in 50 mM PIPES, pH 6.8. The column
was washed with 25-50 ml of the same buffer and eluted
with a linear KCI gradient. Peak fractions were pooled and
concentrated by ultrafiltration and applied to either Sep-
hadex G-75 (1.6cm i.d. x 55cra) or Superose 6 (l.6cm
i.d. x 48 mm). Columns were calibrated using Blue Dextran
2000 and a range of standard proteins.
Enzyme assays
Three activities were monitored: proteolytic, esterolytic
and amidolytic.
(l) Proteolytic. The assay (Kunitz, 1947) contained 5.5 mg
sodium caseinate in a final volume of 1.1 ml of 50 mM
Tris--HCl, pH 8.0. After incubation for 30 rain at 30°C, the
reaction was stopped by addition of 0.5 ml 30% TCA.
Following centrifugation, absorbance of aromatic amino-
acids as solubilized peptides was measured at 280nm.
Elastase was assayed using the elastin-orcein method of
Mandl (1962).
(2) Esterolytic. Trypsin (TRY), chymotrypsin (CHY) and
carboxypeptidase A (CPA) were assayed (Gooding and
Rolseth, 1976; Baker, 1981). Assays contained, in a final
volume of 2 ml 50raM Tris--HCI, pH 8.0; 1 mM BAEE
(TRY at 255 nm, E = 808), 1 mM BTEE in 17% MeOH
(CHY at 256 nm, E = 964) or 1 mM HPLA (CPA at 254 rim,
E = 592). Thiol protease activity was assayed at 30°C using
1 mM BAEE in 0.1 M phosphate, pH6.0 containing
0.5mM DTT and 0.15mM EDTA. The assays measured
the appearance of hydrolyzed product.
(3) Amidolytic. Trypsin, chymotrypsin, elastase, leucine
aminopeptidase (LAP) and carboxypeptidase B (CPB) ac-
tivities were assayed (Gooding and Rolseth, 1976; Baker,
1981; Bieth et al., 1974). Assays contained, in 1.1 ml 50mM
Tris-HC1, pH 8.0; I mM or 0.5 mM D,L-BApNA in 5%
D M F (bovine and grass grub TRY respectively, 1 mM
BTpNA in 20% D M F (CHY), I mM SApNA in 10%
DMSO or 1 mM LpNA in 10% methanol (LAP). Following
incubation for 10-30 min at 30°C, reactions were terminated
with 0.5 ml 30% acetic acid. Following centrifugation, if
necessary, absorbance of p-nitroanilide in the supernatant
was read at 410 nm (E = 8800 cm-l). Carboxypeptidase B
was assayed as described for esterolytic assays using I mM
HA (254 nm, E = 360).
The titration for carboxypeptidase inhibitor, described in
the legend to Fig. 6, followed the method of Green and
Work (1953).
Polyacrylamide gel electrophoresis
(I) SDS-PAGE. Protein, precipitated from crude gut
extracts or from aliquots of column fractions by addition of
TCA to 12%, was washed thrice with 0.4 ml of cold acetone
before being dissolved in 20 gl of the extracting solution of
Smith and Payne (1984). The samples were heated for
10min at 85°C before analysis on a 15% polyacrylamide
slab gel containing 0.1% SDS (Laemmli, 1970) with both gel
and run buffer concentrations increased 1.5 times (Fling
and Gregerson, 1986). Gels were fixed and stained with
Coomassie Blue as in Gardiner et aL (1986).
(2) Activity gel. Crude extracts for electrophoresis were
prepared by homogenizing 10 larval guts in 400 #1 of 0.12 M
Tris-PO4, pH 6.8, centrifuging at 20,000g, 5 min and dilut-
ing. the resulting supernatant 1:I with a loading buffer
containing 0.12M Tris-PO4, pH6.8, 50% glycerol and
0.02% bromphenol blue. Aliquots (5#1) were used for
electrophoresis. Aliquots (650 #1) from peak DEAE column
fractions were mixed thoroughly with an equal volume of
cold 60% polyethylene glycol 4000, 5 itl of 10 mg/ml BSA
added, the samples were mixed again and then kept at 4°C
overnight. After centrifugation, 20,000 g, 15 min, the super-
natant was discarded. The pellet was redissolved in 40 #1 of
loading buffer and the whole sample analyzed in a 7.5%
polyacrylamide gel.
To observe trypsin activity, a piece of Whatmann 3 MM
filter paper was impregnated with a solution of I% Noble
Agar in 50 mM Tris-HCl, pH 8.5, cooled and air dried
before being flooded with a solution of 1 mM BANA in
50mM Tris-HC1, pH 8.5 and 5% DMF. This substrate
overlay was drained and incubated in contact with the
electrophoresed gel at 30°C for 30 min. Blue bands were
then visualized against a pink background by incubating the
overlay for about 10 min in 100 ml of 0.33 M NaAc, pH 4.5
containing 200 mg MgCI2, 130 mg Fast Black K salt and
5 ml of Triton X-100. The overlay was briefly rinsed in
0.33 M NaAc, pH 4.5 prior to photography.
Data analysis
Molecular weights of gut proteases by gel chro-
matography were calculated from a linear regression of log
MW against Kay for the standard proteins.
Kinetic parameters for the ester substrates HPLA and
BAEE were obtained from progress curves using the re-
lationship
p/t = Vmx - Kin(log,[(So - P)/Sol)/t
where t = time, p = A at t, So = total A and Vm~xand K~ are
the kinetic parameters. The data were fitted by linear
regression.
Kinetic parameters for p-nitroanilide substrates were
obtained using a computer program which fitted the data by
nonlinear regression methods to one of three equations.
Data for LAP were fitted to the Michaelis-Menten equa-
 Gut proteases of grass grub 223

tion. The data for bovine trypsin were fitted to the equation
v = v~x/(2 + K~/S)
and those for grass grub trypsin to
v = V,~,/(2 + Km/S + 2 S / K . )
where v = observed rate, S = substrate concentration, V=,x
and Kmare the Michaelis-Menten kinetic parameters and K,
is a substrate inhibition constant. These equations imply the
following assumptions: (1) that D,L-BApNA consists of 50%
L-BApNA as substrate and 50% D-BApNA as competitive
inhibitor; (2) that K~ (L-BApNA) equals/~ (D-BApNA); and
(3) in the case of gras grub trypsin only, that both D- and
L-BApNA bind at a second inhibitory site with the same
inhibition constant Kn.
The activity-inhibitor titration curve for CPA was ana-
lyzed by the method of Green and Work (1953) assuming,
(l) a stoichiometry of I:1 for protease:protease inhibitor
binding; and (2) that the carboxypeptidase-inhibitor com-
plex is catalytically inactive (Ryan et al., 1974). Solving the
equation for a single step binding mechanism plus the
relevant conservation equations then leads to the following
relationship:
vi/v o = (E -- I -- K + [(I -- E + K) 2 + 4EK]t/2)/2E
where vdv o is the fractional activity, I is the known total
inhibitor concentration, E the equivalence point (the in-
hibitor concentration equal to the protease concentration)
and K the equilibrium dissociation constant (Kd) for the
binary complex. The unknown parameter values were calcu-
lated by nonlinear regression.
RESULTS
P r o t e a s e activity in crude e x t r a c t s
Estimates based on the relative activities toward
amide and ester substrates in crude extracts indicated
high trypsin and low chymotrypsin and elastase
activities, with intermediate levels of leucine amino-
peptidase and carboxypeptidase A and B (Table 1).
These activities are predominantly in the midgut
region rather than the large expanded darker-
coloured hindgut or fermentation sac (Allison, 1969;
Bauchop and Clarke, 1975) (Table 1). The protease
activity is mainly present in the lumen rather than the
gut walls (Table 2).
Little effect on any protease activity resulted from
varying the pH (6.0-8.0) or ionic strength (0-0.15 M
NaCl) of the extraction buffer or by addition of
fl-mercaptoethanol (0-10 raM), PVPP (0-5%),
E D T A (0-5 mM) or passing the extract through a
Sephadex G25 column. Consequently, we believe the
data presented (Table l) are a true reflection of the
relative activities of soluble proteases in the larval
gut.
Purification
The proteinase activity was resolved into four
caseinolytic peaks by D E A E chromatography (Fig.
1). Three caseinolytic peaks appeared to closely coin-
cide with CPA and the two peaks of trypsin activity.
The fourth peak, that followed the CPA peak (Fig.
l, peak II), was not further characterized. Pooled
peak fractions were used for subsequent purification
and characterization of the major activities present.
The two peaks of trypsin activity were labelled TRY-
1 and TRY-2 (in the order of elution from the
column).
S o m e p r o p e r t i e s o f larval g u t p r o t e a s e s
Measurements of midgut pH indicate a value of
pH 7.0-7.1 in both fresh gut contents and homoge-
nized aqueous extracts of frozen dissected gut. The
pH response of each major protease activity detected
in grass grub larval gut extracts is described in Fig.
2. Although the pH optima of all proteases is above
the measured pH of the gut, they all retain substantial
activity at this pH.
Substrate saturation-velocity curves indicative of
Michaelis-Menten kinetics were not obtained for
TRY-1 and TRY-2 when D,L-BApNA was the sub-

Table 1. Localizationof major grass grub larval gut proteases

Midgut Hindgut
Enzyme Substrate (nmol min- i larva- ~) (nmol min- l larva- i )
Trypsin BAEE 14,700 80
BApNA 179 2.6
Chymotrypsin BTEE 930 8
BTpNA 2.4 0.1
Elastase SApNA 6.3 ND*
Lcucine aminopeptidas¢ LpNA 31 ND
Carboxypeptidas¢ A HPLA 1460 70
Carboxypeptidase B HA 970 ND
Proteinase Casein 0.107t 0.002
*ND, not determined.
"['280nm absorbance units min- ~ larva- ~.
The crude hindgut extract was prepared identicallyto the midgut extract as described in the Methods.

Table 2. Midgut compartmentalizationof major grass grub larval gut proteasvs

Intact gut Gut contents Gut walls
Enzyme Substrate (nmol min -I larva -I) (nmol min -I larva -I ) (nmol min -I larva -I)
Trypsin BApNA 141 88 31
Chymotrypsin BTpNA 2.1 1.9 0.1
Proteinase Casein 0.083" 0.063 0.018
*280 nm absorbance units min-i larva-~.
A crude extract was prepared from I freshly dissected intact larval gut as usual. Gut contents were obtained from 3 freshly
dissected intact guts using a pasteur pipette, added to I ml Tris-HCI, pH 8.0 and centrifuged. Gut walls were prepared
by cutting 3 guts open, removing contents and gently washing walls in distilled water at 4°C. The tissues were
homogenized and centrifuged.
224 J . T . C~IST~LL~ et al.

DEAE CHROMATOGRAPHY OF CRUDE EXTRACT

i

80 I- LEGEND /OR
701- e TRY Iii : ~
6OF- o CHY xlO ~ III d,
1 • LAp ,~,,® • rlv •
50i- • CPA x l ~_ ~ i~ • "~
oq
=,
o cAsEIN ,~ ~, ~'~.,' ~,,~ ',
o
~o~- ,',,~I~ r ',
>
o
or .,'~i .'' ~ ''~ ";
~'o ~o ~o ~o ~o ~'o
FRACTION NUMBER

Fig. 1. Approximately 80 larval guts were prepared as a crude extract in 19 m125 mM PIPES, pH 6.8 and
applied to a DEAE column (16 mm i.d. x 118 ram). A linear KCI gradient (0-0.45 M, fractions 1~50) was
used to elute the proteins, at 4°C and 25 ml h-L Fraction size was 12.5 ml. Much of the protein and A~0
absorbing material eluted in the void volume, but only trace amounts of proteinase activities (data not
shown). Recoveries were all close to 100%. The Roman numerals identify the four peaks of caseinolytic
activity.

strate; the curves decreased at high substrate concen-
trations (Fig. 3). However, when the simplest possible
model for substrate inhibition was combined with
that for competitive inhibition to account for the
presence of D-BApNA as described in the Methods,
an entirely satisfactory fit was obtained. The kinetic
parameters Km and k=t obtained from this model for
TRY-1 and TRY-2 were not significantly different
(Table 3). TRY-I and TRY-2 have a lower Km and
higher k~t than bovine trypsin for both synthetic
substrates (Table 3), indicating a 15 to 20-fold in-
crease in catalytic efficiency as measured by the
specificity constant (k=,/Km) (Fersht, 1974; Brockle-
burst, 1977).
The data for the substrate saturation-velocity
curves for LAP and CPA could be fitted using
Michaelis-Menten kinetics and the parameters are
shown in Table 3.
The molecular weights of the major larval gut
proteases were determined (Table 4). The values from
SDS-PAGE are lower but in reasonable agreement
with those obtained by gel permeation chro-
matography.
The similarity of their kinetic parameters and
molecular weights suggested that TRY-1 and TRY-2
might be isoenzymes or single species modified post
translationally and the presence of multiple bands
following SDS-PAGE analysis of fractions purified

EFFECT OF pH ON PROTEASE ACTIVITIES

=

.A
120
!0 t" LEGEND ,g' • "a,

~ )0
100
I0 I-
so
•
•
•
TRY-2
LAP
CPA
o CASEIN /(,'"
'
~/
/" ~
,,,~ .e- - • - -e
- - ~ 7 ~,

|0 I-
eo ~ / , ~

,~ 40
==
2~0
0 I- ,E( ~ '""~

Ot-

pH

Fig. 2. Pooled peak fractions from DEAE chromatography were used as enzyme sources for TRY-2, LAP
and CPA. Caseinolytic activity was measured using a crude gut extract. Buffers were prepared as follows:
pH 4.5, I00 mM Na acetate: pH 5.0, I00 mM Na acetate; pH 5.5, 50 mM Na acetate-50 mM MES; pH 6.0,
100mM MES; pH6.5, 50mM MES--50mM MOPS; pH 7.0, 100mM MOPS; pHT.5, 50raM
MOPS-50 mM Tricine; pH 8.0, 8.5, 100 mM Tricine; pH 9.0, 9.5, I0.0, I00 mM Triethanolamine. The
data points represent duplicate or quadruplicate measurements. The effect of pH on the Michaelis constant
was not determined. The protease activity units are arbitrary.
 Gut proteases of grass grub 225

EFFECT OF SUBSTRATE ON TWO TRYPSINS

i i " 't i i

35i-
~" LEGEND
:~ 3°I • TRY-2
g 25~ o BOVINE / ' -

OF-
I I
; 5 ; 1.5
[L-eApNAI mM

Fig. 3. Grass grub TRY-2 assays (quadruplicate) and bovine trypsin (10 pg) (duplicate) were carried out
in 50 mM Tris-HCl, pH 8.0 at 30°C in various concentrations of DL-BApNA. The concentrations of
L-BApNA were calculated and data analyzed as described in the Methods section. The Michaelis
parameters are given in Table 3 and the lines are those fitted.

on Sephadex G-75 (Fig. 4) indicated that additional
forms might be present. When fractions were ana-
lyzed by P A G E under non-denaturing conditions
(Fig. 5), four distinct bands of similar activity level
were detected in crude extracts. The most rapidly
migrating form of these comigrated with T R Y - l ,
consistent with its early elution from D E A E . The
T R Y - 2 fraction contained the three slower moving
forms. These were unresolved by D E A E chro-
matography, although the width of the T R Y - 2 peak
and the presence o f shoulders in the profile suggest
partial resolution may have occurred.
Inhibition studies
Protease inhibitors of known ranges of specificity
were used (1) to assess the contribution of each
enzyme to proteolysis in crude extracts; and (2) to
further characterize the protease class to which each
enzyme belonged.
The effect o f a range of inhibitors with specificities
toward different proteinase classes is shown in Table
5. Proteolysis and B A p N A hydrolysis by crude ex-
tracts was not stimulated by reducing agents nor
inhibited by thiol inactivating compounds a,t p H 8.
These results strongly suggest that thiol proteinases
are absent from gut extracts. Some residual pro-
teolytic activity remains in gut extracts at p H 6.0
(Fig. 2), the p H optimum o f c o m m o n thiol pro-
teinases (e.g. papain, bromelain and ficin). However,
no thiol proteinase activity is present at p H 6.0 in gut
extracts; proteinase activity is due to tryptic digestion
(Table 6).
Metal ion chelating agents inhibited crude extract
proteolytic activity by 10% or less (Table 5), sug-
gesting metallocndopeptidases are not a major con-
stituent of gut extracts. E D T A did not inhibit the gut
LAP, a metalloexopeptidase (Table 6). This enzyme
was inhibited by heavy metals, but not by P M S F ,

Table 3. Kinetic parameters for major gut proteases and bovine trypsin
Kin* K, k~=
Enzyme Substrate OaM or raM) (raM) (s -I)
TRY-1 BApNA 0.37 + 0.08 0.94 + 0.22 NDt
TRY-2 BApNA 0.27 4- 0.04 0.79 ± 0.12 5.7 :t: 0.5:~
Bovine trypsin BApNA 2.53 ± 0.41 -- 1.3 ± 0.1
TRY-2 BApNA 0.50 ± 0.08 0.27 + 0.05 ND~
Bovine trypsin BApNA 2.13 ± 0.12 -- 1.9 ± 0.1
TRY-2 BAEE 1.50 ± 0.09 -- 57. I _+0.43
Bovine trypsin BAEE 10.9 ± 1.0 -- 23.6 + 0.4
LAP LpNA 0.78 ± 0.02 -- ND
CPA HPLA 28.0 ± 1.1 -- 6.8 ±0.2¶
*Ester substrate (BAEEand HPLA) valuesexpressed in/~M, p-nitroanilide substrate
values in raM.
"['Not determined, gut proteases only partially purified.
~/Calculation based on protein trypsin inhibitor binding stoichiometries(Christeller
and Shaw, 1989).
§Specificactivity 0amol BApNA mg protein-i rain-i) of TRY-2 was 2.3-fold that
of bovine trypsin (data not shown).
¶Data calculated from carboxypeptidas¢ inhibitor binding (Fig. 6).
All assays were carried oat and data was analysed as described in Methods section.
The initial portion of the progress curve for TRY-2 was fincar, commencingat
0.35raM BAEE (230 x K=); hence substra~ inhibition, as seen with D,I.-
BApNA, was not observed.
226 J. T. CHRISTELLER et al.

Table 4. Molecular weights of major gut proteases
Enzyme Method Molecular weight
TRY- I Sephadex-G75 28,500 + 1000
SDS-PAGE 22,900 + 970
TRY-2 Sephadex-G75 28,500 + 1000
SDS-PAGE 23,560 __ 1230
CPA Superose 6 42,800 + 1300
LAP Superose 6 96,400 + 1300
SDS-PAGE 90,960 + 1020
The gel permeation columns were calibrated using the
following standard proteins: Rhodospirillum rubrum
ribulose bisphosphate carboxylase dimer, oval-
bumin, bovine serum albumin, carbonic anhydrase,
trypsin, cytochrome c and insulin. Columns were
equilibrated in 5 0 m M Tris-HCl, pH 8.0, samples
loaded as 0.5 ml aliquots and columns eluted at
0.Sml min -I (Sephadex-G75) and 1.0ml m i n - '
(Superose 6). Proteins were detected by enzymatic
activity, absorbance at 280, 660 (Blue Dextran) or
554nm (cytochrome c). Analysis by S D S - P A G E
was performed as described in Methods. The TRY-
1 and TRY-2 samples were aliquots of Sephadex-
G75 peak fractions. Good fits to linear regressions
were obtained. For the Sephadex-G75 column,
n = 5, R 2 = 0.995; for the Superose 6 column, n = 7,
R 2 = 0.994; and for SDS-PAGE, n = 9, R 2 = 0.985
and 0.995.
results typical of LAP from other sources (Delange
and Smith, 1971). Casein hydrolysis by crude extracts
was inhibited 20% by 0.2 mM Cd 2+ (Table 5), at
which concentration LAP is completely inhibited
(Table 6).
Four different families of specific serine proteinase
inhibitors (Kazal--CEOM, Animal Kunitz--
Aprotinin, Plant Kunitz--SBTI and Bowman-
Birk--SBBI, CPTI, LBTI) (Laskowski and Kato,
1980) almost totally inhibited BApNA hydrolysis in
both crude and partially purified extracts (Table 5).
Casein hydrolysis was inhibited 60 and 80%, re-
spectively, in these fractions under identical condi-
tions. The results confirm earlier data that most of the
gut proteinase activity derives from a mixture of
closely related serine proteinases of the trypsin type.
Carboxypeptidase A activity was titrated with a
specific proteinaceous inhibitor from potato tubers
(PCPI) (Fig. 6). The data were fitted to the equation
described in the methods from which both the dis-
sociation constant Kd = (6.65 + 0.58) x 10 - s M and
the amount of enzyme present in the assay
E = (6.7 _ 3.3) x 10-s M were determined. The latter
value was used to calculate kca t (Table 3).
PCPI inhibited casein hydrolysis in crude extracts
by 20% (Table 5) at a concentration where CPA was
inhibited 80% (Fig. 6).
DISCUSSION
Digestive physiology
It is probable that the larval midgut is the source
of the proteases, although microbial sources could
not be ruled out since the activity associated with the
gut wall may represent enzyme bound to the external
membranes of the walls rather than internal pools
prior to secretion. However, although the hindgut
contains most of the bacteria and protozoa (Bauchop
and Clarke, 1975), soluble proteases are virtually
absent there.
Because the relative amounts of different proteases
in the midgut and the hindgut are similar, it is
probable the enzyme activity in the hindgut repre-
sents proteases secreted in the midgut and carded on
with the digesta. Since the levels are low in the
hindgut, either retention in the midgut occurs or the
proteases are rapidly degraded in the hindgut.
The midgut contains a high level of protease
activity, which when further examined contains both

Table 5. Effect of inhibitors on casein and BApNA hydrolysis

Casein hydrolysis BApNA hydrolysis
Enzyme fraction Inhibitor Concentration (% control) (% control)
Crude extract DTT* 5 mM 100 --
IAA* 1 mM 99 96
NEM* 1 mM 97 99
DTNB* 1 mM 100 95
EDTA'[" 1 mM 98 --
2,2'-bipyridylt 5 mM 92 97
1,10 phenanthroline~" 10 mM 89 103
PMSF:~ I 0 mM 49 73
TLCK~: 0.1 mM 67 6
CEOM§ 9 ,aM 47 19
SBTI§ 12 ,a M 65 0
SBBI§ 31 ,aM 39 5
CPTI§ 29 ,a M 36 0
LBTI§ 28 ,a M 45 5
Aprotinin§ 38 ,a M 36 0
Cd2+¶ 0.3 mM 82 --
PCPI** 2.2 ,a M 84 --

DEAE-TRY-2 PMSF 10 mM 19 3
Antipain 75 ,a M 24 4
SBTI 5 ,a M 17 3
LBTI I 1 ,a M 17 2
*Thiol protcinas¢ reagent (DTT can activate oxidized thiols).
~'Metalloproteinase inhibitor.
:~Scrine proteinase inhibitor, but also may attack thiol proteinases.
§Serine proteinase inhibitor.
¶Leucine aminopeptidas¢ inhibitor.
* *Carboxypvptidase inhibitor.
Enzyme fraction plus inhibitor were incubated for at least 20 rain at p H 8.0, 30°C in 50 mM Tris--HCI prior to
addition of substrate. Ethanol alone, used to solubilize PMSF, 2,2'-bipyridyl, and 1,10 phenthroline; had no effect
on control rates.
1 2 3 15! (4)

ORIGIN
•~ 116
~ 97.4

66

~ 4 5

36

m 29
/ 24

20.1

14.2
FRONT •
GUT 6 20 32 43
~ FRACTION
Fig. 4. Analysis of column fractions by SDS-PAGE. Lanes I and 5, molecular weight standards; lane 2,
LAP; lane 3, TRY-l; lane 4, TRY-2.
Fig. 5. Separation of trypsin activities by gel electrophoresis under non-denaturing conditions. The trypsin
activity was detected after blotting to filter paper from the acrylamide gel as described in the Methods.
Lane 1, crude extract; lanes 2-5 fraction numbers 6, 20, 32 and 43 from DEAE column (Fig. 1).

227
 Gut proteases of grass grub 229

Table 6. Effect of inhibitors on LpNA, BTpNA and BAEE hydrolysis

Enzyme fraction Substrate Inhibitor Concentration % Control
DEAE-LAP LpNA* PMSFS IOmM 102
CdCl,$ 0.2mM 1
CdCI, ImM 0
CUCI, ImM 11
CUCI, 5mM 0
EDTA 5mM 93

DEAE-CHY BTpNA* PMSF IOmM 3

Crude extract BAEEt -DTT/EDTA 0.5/0.15mM 100

IAAn 0.54 mM 92
E-640 13pM 100
Cystatinl/ 0.82 pM 92
SBTI 0.24 uM 11
lp-Nitroanilide substrate assays preincubated (at least 20 min) and assayed at pH 8.0.
tBAEE assays preincubated (at least 5 min) and assayed at pH 6.0. BAEE is hydrolyzed by both
serine and thiol proteinases.
SSerine or thiol proteinase inhibitor.
gHeavy metal inhibitor of LAP.
l]Thiol proteinase inhibitor.
**Serine proteinase inhibitor.
Ethanol and DMSO alone, used to solubilize PMSF and E-64, respectively, had no effect on the
control rates.

endopeptidase (TRY, CHY, elastase and peak II)
and the two major types of digestive exopeptidases
(CPA and CPB and LAP). These, all retaining high
activity at physiological pH, would appear to be
adequate for total protein digestion. Additionally,
TRY-2 showed a distinct preference for proteins
purified from clover roots, hydrolyzing this substrate
at 3 to 5-fold the rates for other proteins (casein,
hemoglobin, gelatin, BSA, gliadin) using the same
concentration and conditions (data not shown).
Despite observations (Murdock et al., 1987; Kitch
and Murdock, 1986; Gatehouse et af., 1985) that thiol
proteinases are widespread in Coleoptera, we could
not detect their presence in grass grub larvae. Even
at optimal acidic pH, no stimulation by thiol com-
pounds nor inhibition by thiol reagents was observed.
The specific thiol proteinase inhibitors E-64 (Hanada
et al., 1978) and cystatin (Barrett et al., 1986) were
also without effect. We also deduce the absence of
metalloproteinases in grass grub larva gut from the
failure of several metal ion chelating agents to inhibit
proteolytic activity. Because the pH response curve
for protein degradation showed little or no activity in
the acidic range, we did not attempt to detect aspartic
acid proteinase type activity in our extracts. The
inhibitor experiments show that about 65% of casein
hydrolytic activity in crude extracts is due to trypsin
activity. The residual activity could be due to the
chymotrypsin and elastase activities, the three exo-
peptidase activities detected and the uncharacterized

INHlBlTlON OF CARBOXYPEPTIDASE A

0 10 20 30 40 50 60 70 60
[PCPI] M x10’

Fig. 6. The peak fraction from DEAE chromatography was used as a source of carboxypeptidase A.
Aliquots (O-12 ~1) of potato tuber carboxypeptidase inhibitor (PCPI) (0.6 mg ml-‘) (MW = 4200) were
mixed with 100 ~1 enzyme, incubated 5 min at 30°C in 1.8 ml total volume 50 mM Tris-HCl, 25 mM NaCl,
pH 8.0; and the reaction initiated with 200 ~1 HPLA (3.35 mg ml-i). Initial velocities, which remained
constant for 10 min at least, were determined and background rates for enzyme plus inhibitor only and
inhibitor plus substrate only were subtracted. Inhibitor concentration was calculated from weight and
stated purity. Individual data points are shown and the line represents the equation fitted with the derived
parameters (see text).
230 J. T. CHmST~LL~et al.
peak II activity. This is particularly true for carboxy-
peptidase since some molecular species of casein are
particularly rich in aromatic amino acids at the
carboxyterminal region (Eigel et al., 1984).
Trypsin
A serine proteinase of the trypsin type is the major
digestive proteinase of grass grub larva midgut. Its
dominant status is assumed from its very high level
detected relative to other activities, its substantial
caseinolytic activity despite casein being a poor tryp-
sin substrate (Reimerdes and Klostermeyer, 1976), its
contribution to casein hydrolysis based on inhibitor
studies, the similarity between pH profiles for
BApNA and casein hydrolysis, its high rate of hydro-
lysis of protein from clover root and its high catalytic
power. The midgut trypsin of third instar larvae
consists of four closely related forms of roughly equal
activity against BANA. The relationship between
these forms is not known. However, the presence of
multiple forms of catalytically active trypsin, pro-
duced through autocatalytic cleavage, has been well
characterized for bovine trypsin (Keil, 1971).
Grass grub trypsins are generally similar to tryp-
sins from other insects. Molecular weights for insect
trypsins range between 17,000-32,000 (Baker, 1981;
Law et al., 1977; Levinsky et al., 1977; Houseman et
al., 1987; Gooding and Rolseth, 1976). Our deter-
minations of 23,000 and 28,000, depending on
method, fall within this range. The pH profile for
TRY-2 is very similar to those of digestive trypsins of
the black carpet beetle larva (Attagenus megatoma,
Dermestidae) (Baker, 1981) and also of adult Diptera
(Houseman et al., 1987; Gooding and Rolseth, 1976).
Kinetic parameters, Km and k~t, of TRY-I and
TRY-2 were also similar to those of black carpet
beetle larvae (Baker, 1981).
Chymotrypsin and elastase
Due to the low level of chymotrypsin and elastase
relative to trypsin (1-5%) these enzymes were not
further characterized. Peak fractions of CHY from
DEAE chromatography lacked activity against tryp-
tic substrates, but were inhibited by PMSF, indi-
cating CHY is likely to be a serine endopeptidase
similar to those of other insects (Baker, 1981). The
presence of elastase activity was confirmed by assays
using the specific substrate, elastin--orcein.
Leucine aminopeptidase
Grass grub larval midgut LAP was characterized
by its substrate specificity and its inhibition charac-
teristics. LpNA hydrolysis, as with LAP from other
sources, was not affected by chemical or pro-
teinaceous trypsin inhibitors, but was inhibited by
the heavy metal ions Cd 2+, Cu 2+, Hg 2+ and Pb 2+
(Delange and Smith, 1971).
The pH optimum of 7.5-8.5 is very similar to that
of other insect digestive LAPs, whereas the molecular
weight of grass grub LAP (97,000) is slightly smaller
than those reported for other insect digestive systems
(100,000-1,500,000) (Baker, 1981; Houseman et al.,
1987; Gooding and Rolseth, 1976). The Km(LpNA)
we determined is similar to that of black carpet beetle
larvae (Baker, 1981).
Carboxypeptidase
The presence of carboxypeptidases A and B was
initially characterized by their substrate specificity.
Levels of the two enzymes were comparable in crude
extracts. Only carboxypeptidase A activity was fur-
ther characterized. The pH optimum for grass grub
CPA is similar to those of dipteran species (Gooding
and Rolseth, 1976; Houseman et al., 1987), although
the molecular weight of 42,800 is larger than the
range of 26,000-30,000 found above. Greater vari-
ation exists among other species. A carboxypeptidase
from the moth larva (Tineola bisselliella, Tineidae)
has a molecular weight of 72,000 (Ward, 1976) and
both bovine pancreatic CPA and CPB of 34,000.
The inhibition constant for PCPI determined for
grass grub CPA (7 x 10-8 M) is similar to those of
bovine CPA (5 x 10 -gM) and porcine CPB
(5 x 10 -8 M) (Ryan et al., 1974).
CONCLUSION
The major digestive proteases of grass grub larvae
have been identified and partially characterized.
We have shown here that trypsin can be considered
the major target in attempts to interfere with protein
digestion in grass grub. We hypothesize that the
expression of a suitable trypsin inhibitor gene in plant
roots would impede grass grub digestion. The range
of trypsin inhibitors is vast (Laskowski and Kato,
1980). In a future study (Christeller and Shaw, 1989)
we will report on an in vitro screening procedure for
serine proteinase inhibitors, which could be used to
select material for in vivo screening.
Acknowledgements--It is a pleasure to acknowledge the
generous girls of CPTI from A. M. R. Gatehouse and V.
Hilder (University of Durham, Durham), of SBBI from D.
E. Foard (Instituto Agronomico, Firenze) and of sodium
caseinate from L. Creamer (N.Z. Dairy Research Institute,
Palmerston North). We thank W. Laing (DSIR, Palmerston
North) and O. Sutherland (DSIR, Auckland) for useful
discussions.
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