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206 Elpidina et al. Archives of Insect Biochemistry and Physiology 48:206–216 (2001)
© 2001 Wiley-Liss, Inc.
Compartmentalization of Proteinases and Amylases
in
Nauphoeta cinerea
Midgut
Elena N. Elpidina,1* Konstantin S. Vinokurov,2 Viktor A. Gromenko,1 Yuliya A. Rudenskaya,3
Yakov E. Dunaevsky,1 and Dmitry P. Zhuzhikov2
1
A.N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow, Russia
2
Department of Entomology, Moscow State University, Moscow, Russia
3
Department of Molecular Biology, Moscow State University, Moscow, Russia
Compartmentalization of proteinases, amylases, and pH in the
midgut of Nauphoeta cinerea Oliv. (Blattoptera:Blaberidae) was
studied in order to understand the organization of protein and
starch digestion. Total proteolytic activity measured with
azocasein was maximal at pH 11.5 both in anterior (AM) and
posterior (PM) halves of the midgut, but the bulk of activity
(67%) was found in PM. Total AM and PM preparations were
fractionated on a Sephadex G-50 column and further analysed
by means of activity electrophoresis and specific inhibitors and
activators. The major activity in PM was classified as an un-
usual SH-dependent proteinase with Mr 24,000 and pH opti-
mum with synthetic substrate BApNA at 10.0. The enzyme was
43-fold activated in the presence of 1 mM DTT, insensitive to
synthetic inhibitors of serine (PMSF, TLCK, TPCK) and cys-
teine (IAA, E-64) proteinases, strongly inhibited by STI, and
displayed four active bands on zymograms. In PM, activities
of trypsin-like, chymotrypsin-like, subtilisin-like, and cysteine
proteinases were observed. Aspartic and metalloproteinases
were not detected. In AM, activity of unusual SH-dependent
proteinase also dominated and activity of chymotrypsin-like
proteinase was observed, but their levels were much lower than
in PM. Distribution of amylase activity, exhibiting an optimum
at pH 6.0, was quite the opposite. The major part of it (67%)
was located in AM. Treatment of amylase preparation with pro-
teinases from AM and PM reduced amylase activity twofold.
Abbreviations used: AM = anterior midgut; BApNA = Nα-
benzoyl-D,L-arginine p-nitroanilide; DTT = dithiotreitol;
DMFA = dimethyl formamide; E-64 = L-trans-epoxysuccinyl-
L-leucylamido(4-guanidino) butane; EDTA = ethylenedi-
aminetetraacetate; GlpFpNA = pyroglutamyl-phenylalanine
p-nitroanilide; Hb = hemoglobin; IAA = iodoacetamide; PM
= posterior midgut; PMSF = phenylmethylsulphonylfluorid;
STI = soybean Kunitz trypsin inhibitor; TCA = trichloroace-
tic acid; TLCK = Nα-tosyl-L-lysine chloromethyl ketone;
TPCK = tosyl-L-phenylalanine chloromethyl ketone; U = unit
of activity; UB = universal buffer; ZAALpNA = benzoyloxy-
carbonyl-alanyl-alanyl-leucine p-nitroanilide.
Grant sponsor: Russian Foundation for Basic Research;
Grant number: 96-04-50782, 99-04-48783.
*Correspondence: Elena N. Elpidina, A.N. Belozersky Insti-
tute of Physico-Chemical Biology, Moscow State University,
Vorobjevy Gory, Moscow 119899, Russia.
E-mail: elp@genebee.msu.su
Received 20 February 2001; Accepted 17 July 2001
Proteinases and Amylases in
N. cinerea
Midgut 207
pH of the midgut contents was 6.0–7.2 in AM, 6.4–7.6 in the first
and 8.8–9.3 in the second halves of PM. Thus, pH in AM is in
good agreement with the optimal pH of amylase, located in this
compartment, but the activity of proteinases, including the abil-
ity to degrade amylase, in such an environment is low. Active
proteolysis takes place in the second half of PM, where pH of
the gut is close to the optimal pH of proteinases. Arch. Insect
Biochem. Physiol. 48:206–216, 2001. © 2001 Wiley-Liss, Inc.
Key words: Nauphoeta cinerea; cockroach; midgut; digestive proteinase;
amylase; midgut pH
INTRODUCTION
The midgut is the main digestive organ of
insects. The recent data indicate that digestive
processes in this part of the gut are arranged both
temporally and spatially. Buffering mechanisms
that are responsible for pH and redox potential,
enzyme activity, absorption of nutrients, and ex-
cretory functions are compartmentalized to spe-
cific regions and allow an efficient sequential
breakdown of food polymers into small utilizable
nutrients. The pattern of midgut digestion corre-
lates well with the phylogenetic position of the
insect. Adaptation to the type of diet consumed
seems to be secondary in evolution (Terra, 1990;
Terra and Ferreira, 1994; Terra et al., 1996).
Cockroaches (Blattoptera), being generally
omnivorous, are among the most primitive and
ancient insects and have retained up to the
present the main characteristics of their neopteran
ancestors. Investigation of the digestive system
of such a successful group is of special interest.
The data on compartmentalization of midgut
pH and digestion in the order Blattoptera are frag-
mentary and contradictory. Luminal pH over a
length of the whole midgut of Periplaneta ameri-
cana (Blattidae) (Greenberg et al., 1970) was
found to be slightly acid, and neutral in Blaberus
craniifer (Blaberidae) (Modder, 1967). In two other
Blaberidae species, Byrsotria fumigata (Fisk and
Rao, 1964) and Leucophaea maderae (Engelmann
and Geraerts, 1980), differentiation of alkaline
posterior midgut (PM) and slightly acidic or less
alkaline anterior midgut (AM) were observed.
Compartmentalization of pH in L. maderae is ac-
companied by spatial organization of proteolysis.
Practically all caseinolytic activity was found in
PM. In P. americana, on the contrary, chymot-
rypsin-like activity predominated in the caeca
(Baumann, 1990). There are no data on the spec-
trum of midgut proteinases in Blattoptera. The
only tested proteinases were trypsin-like in
Nauphoeta cinerea (Blaberidae) (Rao and Fisk,
1965) and chymotrypsin-like in P. americana
(Baumann, 1990). Distribution of amylases and
several other glycosidases in the midgut was stud-
ied only for Panesthia cribrata (Blaberidae). Ac-
tivity of all tested enzymes was predominant in
AM (Scrivener et al., 1989).
The purpose of our work was to analyze spa-
tial organization of pH, digestive proteinases, and
amylases in the midgut of omnivorous cockroach
N. cinerea.
MATERIALS AND METHODS
Materials
Nα-Benzoyl-D,L-arginine p-nitroanilide
(BApNA), dithiotreitol (DTT), Nα-tosyl-L-lysine
chloromethyl ketone (TLCK), and tosyl-L-phenyl-
alanine chloromethyl ketone (TPCK) were obtained
from Fluka (Buchs, Switzerland); phenylmethylsul-
phonylfluorid (PMSF), cytochrom c, and mioglobin
from Serva (Heidelberg, Germany); iodoacetamide
(IAA), L-trans-epoxysuccinyl-L-leucylamido(4-guan-
idino) butane (E-64), ethylenediaminetetraacetate
(EDTA), gelatin, and soluble potato starch from
Sigma (St. Louis, MO); azocasein from Calbiochem
(San Diego, CA); ovalbumin from Wartington (Free-
hold, NJ); a set of indicator dyes from Merck
(Darmstadt, Germany); soybean trypsin inhibitor
(STI) and bovine hemoglobin (Hb) from Reanal
(Budapest, Hungary); pepsin (EC 3.4.23.1) from
Olaine (Riga, Latvia); and Sephadex G-50 from
Pharmacia (Uppsala, Sweden). Pyroglutamyl-phe-
nylalanine p-nitroanilide (GlpFpNA) and benz-
oyloxycarbonyl-alanyl-alanyl-leucine p-nitroanilide
(ZAALpNA) were synthesized in the Department
208 Elpidina et al.
of Protein Chemistry of A.N. Belozersky Institute
of Physico-Chemical Biology, Moscow State Uni-
versity, Moscow, Russia.
Insects
Adult cockroaches, Nauphoeta cinerea Olivier,
were selected from laboratory stock culture and
were fed for 2 weeks on dry ground white bread.
The saliva was collected from cockroaches starved
for two days with an automated pipette after
slight squeezing of the thorax.
pH of Midgut Lumen
A standard set of indicator dyes with over-
lapping regions of color change was used to deter-
mine pH of midgut lumen. The set was composed
of bromphenol blue, methyl red, bromcresol
purple, bromthymol blue, phenol red, cresol red,
thymol blue, cresolphthalein, thymolphthalein,
and alizarin yellow. The cockroaches were held
without food for 1–4 days. Then 5 animals were
fed with 1.5 g of wheat flour soaked with 0.02–
0.1% dye solution in 96% ethanol. The cockroaches
were dissected after 2–24 h and the color of the
AM and PM contents was evaluated.
Total Soluble Protein Preparation
Cockroaches (100–150) were dissected in ice-
cold 125 mM NaCl. The whole gut was removed,
thoroughly washed, and the anterior (AM) and
posterior (PM) halves of the midgut were excised
and homogenized in 0.5 ml of 125 mM NaCl in
glass-glass homogenizer. An equal volume of 10
mM phosphate buffer, pH 7.0, was added and the
extraction mixtures were centrifuged for 15 min
at 5,700g. The supernatants were brought to 70%
saturation with solid ammonium sulfate. The re-
sulting precipitates were collected at 44,000g for
20 min at 4°C and dialyzed against 10 mM phos-
phate buffer, pH 7.0. The dialysates were clari-
fied with centrifugation and NaN3 was added to
the preparations to 0.02% concentration to sup-
press microbial growth.
Gel Filtration Chromatography
Total enzyme preparation from AM or PM
containing 70–80 mg of protein was applied to a
Sephadex G-50 column (2.5 × 120 cm) equilibrated
with 500 mM NaCl in 10 mM phosphate buffer,
pH 7.0, containing 0.02% NaN3. Fractions of 9.0
ml were collected and analyzed for protein con-
tent and activity with different substrates. Active
fractions were pooled, concentrated, and desalted
on Amicon YM3 membranes (Beverly, MA) and
used for further analysis. To assess the molecu-
lar weight (Mr) of enzymes, the column was cali-
brated using cytochrome c (Mr 12,300), mioglobin
(17,800), STI (22,000), pepsin (36,000), and oval-
bumin (45,000).
Assays of Proteolytic Activity and Protein
Determination
Total proteolytic activity at pH 5–12 was
measured with azocasein. One hundred microli-
ters of enzyme was incubated with 200 microli-
ters of 0.5% azocasein solution in 100 mM sodium
acetate-phosphate-borate universal buffer (UB) of
specified pH for 1 h at 37°C. The enzyme reac-
tion was stopped by addition of 300 µl of 12%
trichloroacetic acid (TCA). The mixture was left
to stay for 30 min at 4°C for precipitate forma-
tion and centrifuged at 5,700g for 10 min. An
equal volume of 500 mM NaOH was added to the
supernatant and the absorbance at 440 nm was
measured.
Activity at pH from 3 to 6 was determined
with bovine Hb as substrate. One hundred micro-
liters of enzyme preparation was incubated with
200 µl 1% Hb solution in 100 mM UB of specified
pH for 3 h at 37°C. The reaction was stopped by
the addition of 500 µl of 10% TCA. The absor-
bance of supernatant was measured at 280 nm
after a 30-min stay at 4°C and centrifuging.
Proteolytic activity with synthetic substrates
BApNA, GlpFpNA, and ZAALpNA was measured
spectrophotometrically at 410 nm by p-nitroaniline
release. One hundred microliters of enzyme prepa-
ration was mixed with 680 µl of 100 mM UB of
appropriate pH and incubated with 20 µl of 40
mM solution of BApNA or 20 mM solutions of two
other substrates in dimethyl formamide (DMFA)
for 0.5, 3, and 5 h, respectively, at 37°C. When
ZAALpNA was used as a substrate 150 µl of
DMFA was added instead of the equal volume of
the buffer. Assays for SH-dependent activity were
performed in the presence of 1 mM DTT.
All assays were performed so that the mea-
sured proteolytic activity was proportional to pro-
tein concentration and time. One unit (U) of
proteolytic activity was defined as the amount of
Proteinases and Amylases in
N. cinerea
Midgut 209
enzyme (mg) which caused an increase in optical
density by 0.1 per min in 1 ml of the reaction mix-
ture. Determinations of enzyme activity were
made in 3–5 replicates.
Protein concentration was determined ac-
cording to Lowry et al. (1951) and spectrophoto-
metrically at 280 nm.
Amylase Activity
Assays of amylase activity were performed
as follows. The ammonium sulfate precipitates of
saliva and extracts from AM and PM, obtained as
described above, were dialyzed against 100 mM
acetate buffer with 20 mM NaCl, 0.1 mM CaCl2,
pH 5.7. The clarified dialysates were used for amy-
lase activity determination with soluble potato
starch as a substrate. One hundred microliters of
enzyme preparation were mixed with 900 µl of
0.5% starch solution in 50 mM above mentioned
acetate buffer and incubated for 30 min at 37°C.
The incubation was stopped by addition of 1 ml
of alkaline copper reagent of Felling (2% CuSO4
× 5H2O, 470 mM K, Na-tartrate, 1,880 mM NaOH),
which was prepared prior to experiment. The
mixture was boiled for 10 min, clarified by cen-
trifugation, and the absorbance at 640 nm was
measured. Glucose was used as a standard of
reducing sugars.
All assays were performed under conditions
such that amylase activity was proportional to
protein concentration and time. One unit of amy-
lase activity was defined as the amount of enzyme
that would produce 1 mg of reducing sugars per
min in 1 ml of reaction mixture.
Susceptibility of amylase preparation to pro-
teinases from AM and PM was estimated after
treatment of 2.6 µl of salivary amylase with 197.4
µl of partially purified proteinase preparation in
10 mM phosphate buffer, pH 7.0, for 30 min at
37°C. The residual amylase activity was deter-
mined as described above.
In Gel Assays
Electrophoretic detection of enzyme activities
in N. cinerea midgut was performed by mildly-
denaturing SDS-PAGE (Michaud et al., 1993;
Michaud, 1998). Samples containing 0.05–0.25 gut
equivalents (2–4 µg of protein from PM and 20–
60 µg from AM) prior to electrophoresis were
mixed with an equal volume of nonreducing
sample buffer (125 mM Tris-HCl, 4% SDS, 20%
glycerol, 0.005% bromphenol blue, pH 6.8) and in-
cubated for 30 min at room temperature. Electro-
phoresis was performed in 8% SDS-PAGE slab
according to Laemmli (1970) at 4°C.
Proteinase activity was detected by means
of “overlay” techniques. The gel was washed in
2.5% Triton X-100 for 30 min to allow enzyme
renaturation, rinsed with 50 mM UB, pH 9.0, for
10 min, and placed onto 8% indicator gel without
SDS, pH 9.0, containing 0.025% gelatin as pro-
teinase substrate. The gels were incubated in a
moist chamber for 1–2 h at 37°C and after incu-
bation the indicator gel was stained with Coo-
massie Brilliant Blue (0.15%) in 30% ethanol, 10%
acetic acid solution. Proteinases were visualized
as clear bands on a dark background.
Proteinase inhibitors PMSF (10 mM), TLCK,
TPCK, IAA, or E-64 (all 1 mM) were added to
the enzyme samples before electrophoresis. The
mixtures were incubated for 30 min at room tem-
perature. The effect of DTT was tested after elec-
trophoresis. DTT (1 mM) was added to the rinsing
buffer (50 mM UB, pH 9.0) and indicator gel.
To determine amylase activity, the gel after
the run was washed in 2.5% Triton X-100 for 30
min, rinsed with 100 mM acetate buffer with 20
mM NaCl, 0.1 mM CaCl2, pH 5.7, and incubated
in 1% solution of soluble potato starch in the same
buffer for 30 min at 37°C. After incubation, the
gel was rinsed with water and stained in 1.3% I2,
3% KI solution.
RESULTS
Midgut pH
pH of the caeca and AM lumen was 6.0–7.2,
and was slightly increasing towards the middle
of the midgut, showing 6.4–7.6 in the first half of
PM. Contents of the second half of PM were alka-
line and displayed pH 8.8–9.3.
Comparison of Total AM and PM Proteinase
Preparations
pH levels in AM and PM lumen did not coin-
cide with pH optima of proteolytic activities in this
compartment. Total activities in AM and PM as-
sayed with azocasein (in mildly acidic and alka-
line pH regions) and with Hb (in acidic pH region)
were both maximal in alkaline region at pH 11.5
210 Elpidina et al.
(Fig. 1). Gut proteinase activities per insect
measured with azocasien and specific sub-
strates of trypsin-like and several cysteine
proteinases (BApNA), chymotrypsin-like pro-
teinases (GlpFpNA), and subtilisin-like protein-
ases (ZAALpNA) all predominated in PM (Table
1). Activities with azocasein and BApNA rose
sharply when 1 mM DTT was added to the re-
action mixture. Activity with BApNA in the
presence of DTT was predominant in both
preparations.
Fractionation of Proteolytic Activities
The total enzyme preparations from PM and
AM were fractionated by gel filtration. Activities
with azocasein and BApNA in PM eluted simi-
larly and separated into two peaks, I and II (Fig.
2A). Both activities increased in the presence of
DTT. The same activities in AM were detectable
only with DTT, showing two peaks with BApNA
and one small and broad peak with asocasein (Fig.
3A). In both preparations, a rather low chymot-
rypsin-like activity with GlpFpNA, as compared
to BApNA-hydrolyzing activity, was observed in
peak II (Figs. 2B and 3B). A broad and also faint
peak of subtilisin-like activity with ZAALpNA was
found only in PM. The latter activity apparently
belongs to not less than two enzymes, because dif-
ferent parts of the peak had different responses
to the decrease of pH in the incubation mixture
from 9.0 to 7.0 (Fig. 2C).
The results of purification of the major SH-
dependent activity with BApNA from peak II of
AM and PM by means of gel filtration are sum-
marized in Table 2. Total activity from AM after
fractionation increased almost 1.5-fold, and this
suggests that a factor inhibiting this activity in
the extract dissociated from the enzyme during
gel filtration in the presence of 500 mM NaCl.
Specific activity increased 15.5-fold during this
procedure. The same data on gel filtration of ac-
tivity from PM do not point to an inhibitor exist-
ence. Activities with BApNA in peak II both from
AM and PM were maximal at pH 10.0. The Mr of
these enzymes was 24,000.
Effect of Specific Inhibitors and Activators
Results of the investigation on the effect of
specific inhibitors and activators on two peaks of
PM proteolytic activity are presented in Table 3.
A broad inhibitor of serine proteinases PMSF and
specific inhibitor of trypsin-like proteinases TLCK
inhibited 39 and 77% of peak I activity with
Fig. 1. Effect of pH on the activity of the total proteinase
preparation from the N. cinerea PM, assayed with azocasein
(1) and hemoglobin (2) and from AM, assayed with azocasein
(3), as described in Materials and Methods.
TABLE 1. Comparison of Proteolytic and Amylolytic Activities in the Total Preparations of N. cinerea PM and
AM Against General and Specific Substrates (Mean ± SD)*
AM PM
Substrate Activity per gut (U/gut) Specific activity (U/mg) Activity per gut (U/gut) Specific activity (U/mg)
Azocasein 0.0039 ±0.0021 0.0015 ±0.001 0.0081 ±0.018 0.013 ±0.02
Azocaseina0.083 ±0.022 0.033 ±0.009 0.12 ±0.015 0.18 ±0.021
BApNA 0.016 ±0.004 0.0063 ±0.0015 0.074 ±0.004 0.093 ±0.005
BApNAa0.28 ±0.06 0.11 ±0.023 3.85 ±0.15 4.83 ±0.18
GlpFpNA 0.011 ±0.002 0.0043 ±0.0021 0.054 ±0.004 0.079 ±0.006
ZAALpNA na na 0.046 ±0.0071 0.108 ±0.004
Starch 0.127 ±0.14 2.77 ±0.121 0.064 ±0.017 4.30 ±0.012
*na, negligible activity.
aActivity was detected in the presence of 1 mM DTT.
Proteinases and Amylases in
N. cinerea
Midgut 211
Fig. 2. Gel filtration on a Sephadex G-50 column of the
total enzyme preparation from N. cinerea PM. A: Elution
profiles of protein at 280 nm (1), activity with azocasein at
pH 9.0 (2), activity with BApNA, pH 9.0 (3), activity with
BApNA in the presence of 1 mM DTT, pH 9.0 (4). B: Chy-
motrypsin-like activity with GlpFpNA, pH 9.0. C: Activity
with ZAALpNA, pH 9.0 (1) and pH 7.0 (2).Fig. 2.
Fig. 3. Gel filtration on a Sephadex G-50 column of the to-
tal enzyme preparation from N. cinerea AM. A: Elution pro-
files of protein at 280 nm (1), activity with azocasein in the
presence of 1 mM DTT, pH 9.0 (2), and activity with BApNA
in the presence of 1 mM DTT, pH 9.0 (3). B: Chymotrypsin-
like activity with GlpFpNA, pH 9.0.
212 Elpidina et al.
BApNA, showing that a trypsin-like enzyme con-
tributed to this activity. Cysteine proteinase in-
hibitors IAA and E-64 partially suppressed the
same activity, implying that cysteine proteinase
was also present in peak I. That was confirmed
by the 3.6-fold increase of the activity with DTT.
Activity with BApNA from peak II of PM was 43-
fold activated in the presence of DTT. Neverthe-
less cysteine proteinase inhibitors IAA and E-64
in high concentrations had little effect on this ac-
tivity. BApNA-hydrolyzing activity from peak II
was also almost insensitive to synthetic inhibi-
tors of serine (PMSF) and among them trypsin-
like (TLCK) proteinases, but was strongly (by
85.6%) inhibited by proteinaceous STI. The effect
of EDTA on proteolytic activity is indicative of the
absence of detectable metalloproteinase activity
in both peaks. Aspartic proteinases are also lack-
ing in the preparation because activity in the acid
region was negligible (Fig. 1). Taking all these
facts together, it may be concluded that BApNA-
hydrolyzing activity from peak II does not belong
to any of the four known proteinase classes and
we consider it to be an unusual SH-dependent pro-
teolytic activity. Classification of activity with
GlpFpNA, detected in peak II, as chymotrypsin-
like, was supported by a strong inhibitory effect
of TPCK (89% of inhibition).
Activity Electrophoresis of Proteinases
Zymographic analysis of total proteolytic ac-
tivities when applied to gelatin-containing gels in
equal gut equivalents revealed that only samples
from PM displayed visible activity and the inten-
sity of the bands increased in the presence of DTT
(Fig. 4). Zymograms of the two peaks of proteolytic
activity, obtained after gel filtration of total PM
preparation (Fig. 2A), are presented in Figure 5.
Activity of peak I is displayed by two major bands,
a and b (Fig. 5A). Activity of band a is suppressed
by PMSF and TLCK, and slightly inhibited by
DTT (Fig. 5A–D), indicating that this is a trypsin-
like activity. Activity of band b is insensitive to
serine proteinase inhibitors and activated by DTT
(Fig. 5A–D), and thus it belongs to cysteine pro-
TABLE 2. Purification of Unusual SH-Dependent Proteinase (Peak II) From AM and PM of N. cinerea
AM PM
Total activity Specific activity Purification Total activity Specific activity Purification
Purification stage (U) (U/mg) fold (U) (U/mg) fold
Extract after ammonium 13.9 0.17 1.0 138.4 3.9 1.0
sulfate precipitation
Peak II after gel filtration 19.9 2.7 15.5 81.2 15.3 3.9
on Sephadex G-50
TABLE 3. Effects of Inhibitors and Activators on the
Activity of Partially Purified Proteinases From N.
cinerea PM (Mean ± SD)
Concen- Residual activity
tration (% of the control)
Reagent (mM) Substrate Peak I (Fig. 2) Peak II (Fig. 2)
PMSF 3 BApNA 61.4 ±2.5 89.9 ±2.83
TLCK 1 BApNA 23.1 ±1.8 101.6 ±1.3*
STI 0.01 BApNA 30.4 ±4.27 15.4 ±2.95
IAA 20 BApNA 66.3 ±3.8 90.3 ±1.6
E-64 0.1 BApNA 89.3 ±5.12 111.4 ±2.3*
DTT 1 BApNA 358 ±8.6 4278 ±34.5
DTT 1 Azocasein 309 ±27.1 1234 ±13.7
EDTA 10 Azocasein 109 ±9.9* 91.9 ±5.2*
TPCK 0.5 GlpFpNA nda10.8 ±2.76
*Enzyme activity is insensitive to the effect of the reagent
(P > 0.05).
and, activity was not determined owing to negligible control
activity.
Fig. 4. Zymograms of the total proteinase preparations from
AM and PM of N. cinerea in the absence and presence of
DTT in gelatin-containing gel. (A) AM; (B) AM with DTT; (C)
PM; (D) PM with DTT.
Proteinases and Amylases in
N. cinerea
Midgut 213
teinase. Activity in peak II was displayed by four
major bands, all of which were activated by DTT
and were insensitive to PMSF, TLCK, IAA, and
TPCK; they presumably belong to the unusual SH-
dependent proteinase (Fig. 6A–F). Activities of chy-
motrypsin-like and subtilisin-like proteinases, found
in peak II after gel filtration (Fig. 2B,C), were mi-
nor as compared to BApNA-hydrolyzing, and were
not detectable in this zymogram, especially taking
into account that gelatin contains a few aromatic
amino acids and is not suitable substrate for chy-
motrypsin-like proteinases (White et al., 1978).
Activity in AM after gel filtration became
clearly visible on zymograms only in peak II (Fig.
6G). It was also present in four bands of the same
mobility as in peak II of PM (Fig. 6F), but the
intensity was different in AM and PM prepara-
tions. In PM, the two upper bands were stronger,
and in AM the two upper bands were weaker. All
the bands were DTT-activated and insensitive to
inhibitors of serine and cysteine proteinases, and
so we consider them to belong also to an unusual
SH-dependent proteinase.
Distribution of Amylase Activity and the Effects
of Midgut Proteinases on It
The data presented in Table 1 indicate that
two thirds of total amylase activity in the midgut
of N. cinerea was found in AM. Amylase activity
was maximal at pH 6.0, which is in good agree-
ment with pH of AM contents. Activity electro-
phoresis of extracts from AM, PM, salivary glands,
and saliva revealed that all preparations contain
one major band of the same mobility (Fig. 7).
Investigation of possible effects of midgut pro-
teinases on amylase activity revealed that protein-
ases of peak II from AM and PM reduced it up to
45 and 57%, respectively (Table 4). Proteinases from
peak I in the same experiment were almost inac-
tive. The confirmation of the proteolytic nature of
inhibitory effects on amylase activity was received
in the experiments with heat denaturation of pro-
teinase preparation (70°C, 10 min). After such treat-
ment of proteinases from peak II, the inhibitory
effect on amylase activity was negligible.
DISCUSSION
Then organization of digestion in anterior
and posterior parts of N. cinerea midgut is differ-
ent. Compartmentalization of digestion is based
on a sharp pH gradient and unequal distribution
of proteinases and amylases.
pH in the midgut of N. cinerea increased from
Fig. 5. Effect of specific inhibitors and activators on the ac-
tivity of partially purified proteinase preparation from peak
I (Fig. 2A) of N. cinerea PM in gelatin-containing gel. (A)
peak I PM control; (B) peak I PM + PMSF; (C) peak I PM +
TLCK; (D) peak I PM + DTT.
Fig. 6. Effect of specific inhibitors and activators on the ac-
tivity of partially purified proteinase preparation from peak
II (Fig. 2A) of N. cinerea PM in gelatin-containing gel (A–F).
(A) peak II PM control; (B) peak II PM + PMSF; (C) peak II
PM + TLCK; (D) peak II PM + TPCK; (E) peak II PM + IAA;
(F) peak II PM + DTT; (G) Zymogram of partially purified
proteinase preparation from peak II (Fig. 3A) of N. cinerea
AM in the presence of DTT.
214 Elpidina et al.
slightly acidic in AM to highly alkaline in PM. A
gradient of midgut pH was earlier observed for two
other blaberoid cockroaches, B. fumigata from 6.0–
6.5 in AM to 8.0–8.5 in PM (Fisk and Rao, 1964)
and L. maderae from 8.0 to 9.0–9.5 (Engelmann and
Geraerts, 1980). In the midgut of B. craniifer
nymphs (Blaberidae), alkaline compartments were
not observed (Modder, 1967), but this result is of
doubtful value, because the author did not use in-
dicators with alkaline regions of color change in the
last third of the midgut. In P. americana from
Blattidae, pH was found to be slightly acidic, 6.3–
6.7, along the whole midgut (Greenberg et al., 1970).
The major part of the activity of N. cinerea
digestive proteinases was located in PM. Its pH
optimum was 11.5 with azocasein and 10.0 with
BApNA, which was close to the luminal pH in this
compartment, 8.8–9.3. Hydrolysis of proteinaceous
(gelatin, azocasein) and synthetic substrates was
predominantly due to SH-dependent activities,
represented by unusual SH-dependent and cys-
teine proteinases, which were described for the
first time in Blattoptera. Proteinases like unusual
SH-dependent, which cannot be related to any of
the recognized classes, are often assigned to the
fifth group of so-called unclassified proteinases
(EC 3.4.99) (Barret, 1980; Antonov, 1991). Zymo-
graphic analysis of unusual SH-dependent pro-
teinase revealed four major bands of proteolytic
activity that might be derived from products of a
multigene family. Several genes or electrophoretic
activity bands of particular serine and cysteine
proteinases were observed in gut extracts of cer-
tain coleopteran (Zhu and Baker, 1999; Koiwa et
al., 2000) and lepidopteran (Peterson et al., 1994;
Bown et al., 1997) insects. A noticeable proteolytic
activity in PM of N. cinerea belonged also to
trypsin-like proteinase, while chymotrypsin-like
and subtilisin-like activities were minor and
couldn’t be detected on zymograms. In gel, pro-
teolytic activity in AM became visible only after
separation from an inhibiting factor during gel fil-
tration. It belonged to an unusual SH-dependent
proteinase, which was displayed also by four
zymographic bands, but with different relative in-
tensity in AM and PM. It should be noted that in
vivo proteolytic activity in AM must be much lower
than measured in our experiments at pH 9.0 (close
to its optimal pH) because of the slightly acidic-
neutral pH in this compartment.
Hitherto investigation of proteinase spectrum
and identification of enzymes responsible for the
bulk of digestive proteolytic activity in Blattoptera
was not performed. Engelmann and Geraerts
(1980) resolved at least four caseinolytic activi-
ties in the midgut of L. maderae. They were pro-
duced and predominated in PM and also displayed
highly alkaline pH optimum, but the study lacks
an assessment of mechanistic classes of protein-
ases. Rao and Fisk (1965) just revealed trypsin-
like activity in the midgut of N. cinerea and
investigated its variations with sex and age.
Baumann (1990) purified chymotrypsin-like pro-
teinase from the intestinal system of P. americana
with Mr 25,000, which revealed two active bands
after electrophoresis. The highest caseinolytic and
chymotrypsin-like activity was demonstrated in
caeca, which might be the place of their synthe-
sis, and thus compartmentalization of proteinases
Fig. 7. Activity of the total amylase preparations from N.
cinerea PM (A), AM (B), salivary glands (C), and saliva (D)
in starch-saturated gel.
TABLE 4. Effects of Midgut Proteinases From Peak I
and II of AM (Fig. 3A) and PM (Fig. 2A) on Amylase
Activity of N. cinerea (Mean ± SD)
Residual activity (% of the control)
Native Native Heated
proteinases proteinases proteinases
Material (peak I) (peak II) (peak II)
AM 104.1 ±3.7* 44.5 ±1.2 94.4 ±5.8*
PM 92.3 ±2.91 57.5 ±2.4 91.2 ±8.3*
*Amylase activity is insensitive to the effect of the protein-
ases (P > 0.05).
Proteinases and Amylases in
N. cinerea
Midgut 215
and pH (Greenberg et al., 1970) in Blattidae ap-
pears to be different from blaberoid cockroaches.
Amylase activity was two times higher in AM
of N. cinerea than in PM. The slightly acidic pH
of AM is in good agreement with the pH optimum
of this enzyme. The only known investigation of
amylase compartmentalization in the midgut of
Blattoptera was performed on P. cribrata (Bla-
beridae). Ninety-seven percent of α-amylase as
well as endo-β-1,4-glucanase and β-1,4-glucosidase
activity of the midgut was found in AM, and the
enzymes from salivary glands, foregut, midgut,
and hindgut had similar chromatographic behav-
ior (Scrivener et al., 1989).
An important consequence of spatial separa-
tion of proteinases and amylases in N. cinerea
midgut seems to be the preservation of amylases
and probably other glycosidases, which have the
same distribution in the midgut, from proteolysis
by endogenous enzymes (Table 4). One of the main
physico-chemical mechanisms providing such com-
partmentalization and protection is pH gradient
in the midgut. Its existence allows for a reduc-
tion in AM of proteolytic activity exhibiting a high
pH optimum. Another possible mechanism of pro-
tection may be the existence in AM of an inhibit-
ing factor active against unusual SH-dependent
proteinase. The protection of glycosidases against
proteolysis is particularly important considering
that portions of meals can persist in the crop for
24 h and are released into the midgut only in
“manageable” portions (Modder, 1967). Preserva-
tion of their activity over a long period of time
would enable the organism to efficiently utilize
food resources with a low metabolic expenditure.
Based on our findings and those of others, it
can be assumed that the above-described type of
midgut compartmentalization is characteristic of
cockroaches from the phylogenetically latest fam-
ily Blaberidae (McKittrick, 1964). Midgut pH and
proteinase distribution of cockroaches from the
primary family Blattidae seem to be different from
that of Blaberidae used in our study.
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