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Quantitative determination of trypsinlike and chymotrypsinlike enzymes in insects.

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Archives of Insect Biochemistry and Physiology 8249-260 (1988)
Quantitative Determination of Trypsinlike and
Chymotrypsinlike Enzymes in Insects
Dov Borovsky and Yosef Schlein
Institute of Food and Agricultural Sciences, University of Florida, Florida Medical Entomology
hboratoy,Vero Beach (D.B.); Department of Parasitology, The Hebrew University-Hadassah
Medical School, Jerusalem (y.S.)
A quantitative and highly specific assay for the determination of trypsinlike
and chymotrypsinlike enzymes in insects has been developed. The assay is
based on the specific binding of [1,3-3H]diisopropylfluorophosphate t o
trypsinlike and chymotrypsinlike enzymes. Trypsinlike enzymes can be
determined specifically in the presence of 10 m M TPCK (tosylarnide-2phenylethyl chloromethyl ketone; chymotrypsin inhibitor) and chymotrypsinlike enzymes can be determined in the presence of 10 m M TLCK
(tosyl-L-lysine chloromethyl ketone HCI; trypsin inhibitor). The assay can
easily detect 65 ng of either trypsinlike or chymotrypsinlike enzymes in
midgut homogenates or whole extracts of many insect species. Using this
assay, we have determined the amount of trypsinlike equivalents in
Phlebotomus papatasi, Pediculus humanus, Stomoxys calcitrans, Musca
domestica, Leishmania major promastigotes, Aedes aegypti, Culex nigripalpus,
Culex quinquefasciatus, and Culicoides variipennis. No trypsinlike equivalents
were found i n Rhodnius prolixus or Ornithodoros moubata. The assay is
useful for comparative studies and can be expanded for use as an electrophoretic fluorographic tool in the study of trypsinlike and chymotrypsinlike
isozymes.
Key words: DFP, DIP derivatives, electrophoresis, fluorography
INTRODUCTION
Trypsinlike and chymotrypsinlike enzymes are the primary proteolytic
enzymes found in the midgut of blood-sucking insects. These enzymes play
Acknowledgments: This work was partially supported by USDA research grant CRCR-1-2394
to D.B. and by the project on Epidemiology and Control of Vector-Borne Diseases in Israel,
REP-NIH-NIAID-AI-1266, AID-CDR project C5-136, and by the UNDPNorld B a n W H O Special
Programme for Research and Training in Tropical Diseases. We gratefully acknowledge the
Lady Davis foundation for supporting D.B. as a Visiting Professor.
Institute of Food and Agricultural Sciences, University of Florida Experiment Station Journal
Series No. 8900.
Received March 31,1988; accepted May 23,1988.
Address reprint requests to Dr. Dov Borovsky, IFAS-University of Florida, Florida Medical
Entomology Laboratory, 200 9th Street S.E., Vero Beach, FL 32962.
0 1988 Alan R. Liss,
Inc.
250
Borovsky and Schlein
an important role in the digestion of the blood meal, in egg development,
and in parasite survival [l-61. In order to study their synthesis and regulation, it is necessary to be able to quantlfy both trypsinlike and chymotrypsinlike enzyme production in the midgut and other tissues of blood-sucking
insects. To quantify these enzymes one can measure either activity, which
does not determine the absolute concentration, or develop a radioimmunoassay as was done for ecdysone and JH* [7-91. Although radioimmunoassay is
very specific and sensitive, different antibodies must be raised for the respective trypsinlike and chymotrypsinlike enzymes found in different insects.
Therefore, radioimmunoassay is not a practical approach for general determination of trypsinlike and chymotrypsinlike enzymes in insects.
The conversion of [1,3-3H]DFP into [1,3-3H]DIP-trypsinand chymotrypsinlike derivatives is specific and a convenient way to tag serine esterases
covalently by phosphorylation of serine residue at the active site of the
enzyme [6,10,11]. Using this technique, Graf and Briegel [El and Borovsky
and Schlein [6] reported that several [l,3-3H]DIP-trypsinlikeisozymes were
synthesized in the midgut of the female Aedes aegypfi and Phlebofomus papafasi. The purpose of this study was to utilize the highly specific conversion
of [1,3-3H]DFP into [1,3-3H]DIP-trypsinlike or chymotrypsinlike derivatives
in order to develop a sensitive, quantitative assay for determination of trypsinlike and chymotrypsinlike enzymes in insects.
MATERIALS AND METHODS
Experimental Animals
Larvae of A. aegypfi, Culex nigripalpus, and Culex quinquefasciafus were
reared at 26°C on a diet of brewer's yeast, lactalbumin, and O.K. Feed lab
chow (l:l:l), under a LD 16:8 cycle. Adults were fed on 10% sucrose or on
chicken blood and females were used 3-5 days after emergence. Adults of
Phleb. papafasi from a colony originally caught in the Jordan Valley were kept
at 28 1°C at 80% relative humidity and fed on 20% sucrose or on blood of
baby mice. Adult Rhodnius prolixus and Ornifhodorosmoubafa were a gift from
Dr. D. Ben Yakir of the Hebrew University of Jerusalem and were fed on
human and rabbit blood, respectively. Adult Pediculus humnus were a gift of
Dr. Kostas Mucuoglu of the Hebrew University of Jerusalem and were fed
mouse blood. Adult stable flies Sfornoxys calcifrans were obtained from Dr. Y.
Braverman of the Kimron Veterinary Institute, Israel, and were blood-fed on
a guinea pig. Adult house flies Musca domesfica were fed sugar or yeast.
Leishmania major LRC-L 137, a Jordan Valley isolate, was grown at 28°C on
NNN medium for 3-5 days. Promastigotes were harvested, washed 3 times
in phosphate-buffered saline, pH 7.4, and brought to a concentration of lo9/
ml. Adult Culicoides variipennis were obtained from Dr. R.H. Baker of the
*Abbreviations: DFP = diisopropylfluorophosphate;DIP = diisopropylphosphoryl; DMSO
= dimethylsulfoxide; J H = juvenile hormone; PAGE = polyacrylamide gel electrophoresis;
PPO = 2,5 diphenyloxazole; TCA = trichloroacetic acid; TLCK = tosyl-L-lysine chloromethyl
ketone HCI; TPCK = tosylamide-2-phenylethyl ketone.
Trypsinlike and Chymotrypsinlike Enzymes
251
Florida Medical Entomology Laboratory, and were membrane-fed on cow
blood.
Preparation of Proteolytic Enzymes
Groups of insects (5 per group) were homogenized with a glass homogenizer in 50 mM TRIS-HCl buffer containing 0.1 M CaC12, pH 7.9, or they
were dissected under a microscope and the posterior midguts were removed,
washed in saline, and homogenized as above. The homogenate was centrifuged for 5 min at 4°C at 10,OOOg. Supernatants were collected and stored at
-20°C. Leishmania major cells were centrifuged in the cold at 6,0008 for 20
min. The precipitated cells were collected, resuspended in 50 mM TRIS-HCl
buffer, pH 7.9, containing 0.1 M CaCl2, and broken by repeatedly freezing
and thawing in liquid nitrogen (at least 3 times). The suspension was then
centrifuged at 4°C at 10,OOOg for 20 min and the supernatant was collected
and stored at -20°C.
Preparation of Trypsin and Chymotrypsin for Calibration Curves
Porcine pancreatic trypsin type IX (Sigma, St. Louis, MO), was further
purified by ion-exchange chromatography and crystallized [l3]. The enzyme
was essentially free of chymotrypsin activity and at least 95% pure. A stock
solution of 1mglml was stored at 4°C in 1mM HCl and was stable for several
weeks.
Bovine pancreas type I1 chymotrypsin (Sigma) was further purified by
cation-exchange chromatography and crystallized 3 times [l3]. The enzyme
was free of trypsin contamination and at least 90-95% pure. A stock solution
of 1mglml was stored at 4°C in 1mM HCl and was stable for several weeks.
Determination of [1,3-3H]DIP-Trypsinlike and Chymotrypsinlike Derivatives
[1,3-3H]DFP (5 pCi in 1pl, specific activity 35 Cilmmol, Amersham) was
incubated with several dilutions of porcine pancrease trypsin (0-5 pg) and
bovine pancreas chymotrypsin (0-5 pg) or with insect homogenates for 18 h
at 4°C in 0.1 ml of 50 mM TRIS-HCl, 0.1 M CaC12 buffer, pH 7.9, containing
8 mh4 TPCK (chymotrypsin inhibitor) or 8 mM TLCK (trypsin inhibitor). At
these concentrations of TPCK and TLCK, inhibition of chymotrypsin or
trypsin activity was complete in analyzed samples. Following incubation, the
[1,3-3H]DIP-trypsinand chymotrypsinlike derivatives were assayed by pipetting aliquots (20-50 p1) onto squares of filter paper (2 x 2 cm) and washing
at 4°C in 10% TCA for 15 min, then twice in 5% TCA for 15 min, followed by
a 5-min wash in absolute ethanol. The papers were dried at 50°C and counted
in a liquid scintillation counter. Calibration curves with trypsin and chymotrypsin were constructed by plotting radioactivity (cpm) of [1,3-3H]DIPderivatives against trypsin or chymotrypsin concentrations in pgl0.l ml.
Concentrations of insect trypsinlike and chymotrypsinlike enzymes were
read directly from the calibration curves or calculated from the slopes and
are expressed as trypsin or chymotrypsin equivalents.
252
Borovsky and Schlein
Polyacrylamide Gel Electrophoresis and Fluorography
PAGE of [1,3-3H]DIP-trypsinlike derivatives was run as described by Borovsky and Schlein [6] and Borovsky [14] in a modified Leammli [l5] on
native slab gels (1mm thick, 15 cm long). The stacking gel was 3% (wlv)
polyacrylamide and 0.125 M TRIS-HC1, pH 6.8, and the separating gel was
10% (wlv) polyacrylamide and 0.375 M WS-HC1, pH 8.8. Samples applied
to the gels contained homogenates of whole insects (1-4 equivalents) or
homogenates of midguts (1-4 equivalents). Gels were stained with 0.1% (wl
v) Coomassie brilliant blue R-250 in a 1:7:6 mixture of acetic acid, methanol,
and water for 30-60 min. Gels were destained for 24 h in 7% acetic acid
containing 5% methanol.
For fluorography, gels were soaked in 100 ml DMSO for 30 min, which
was replaced with fresh DMSO for 30 min more and then with 22% PPO in
DMSO for 3 h. Gels were rinsed and soaked in water for an additional 30
min, dried at 60°C under vacuum in a gel drier (Bio-Rad, Richmond, CA),
and exposed to X-ray film for 1-4 days at -70°C.
RESULTS
Production of Calibration Curves for [1,3-3H]DIP-Trypsinand Chymotrypsin
Derivatives
To find out whether there is a linear relationship between the conversion
of [1,3-3H]DFP into [1,3-3H]DIP-trypsinand chymotrypsin derivatives, different concentrations of trypsin and chymotrypsin (0-6 pg) were incubated in
0.1 ml of 50 mM TlUS-HCl buffer, pH 7.9, containing 10 mM CaC12 for 18 h
at 4°C in the presence of 5 pCi [1,3-3H]DFP. Following incubation, the
concentrations of trypsin and chymotrypsin (pglO.1 ml) were plotted against
the radioactivity (cpm) of [1,3-3H]DIP derivatives (Figs. 1, 2). Both curves
were linear down to 65 ng and up to 6.0 pg for both enzymes (Figs. 1, 2).
Additions of 5 mM or 10 mM TLCK (trypsin inhibitor) caused 93% and 97%
inhibition in the synthesis of [1,3-3H]DIP-trypsin derivatives, respectively
(Fig. 1).On the other hand, TPCK (chymotrypsin inhibitor) did not cause
any inhibition in the synthesis of [1,3-3H]DIP-trypsin derivatives (results not
shown). Similar results were obtained when 5 mM or 10 mM TPCK (chymotrypsin inhibitor) was added to the reaction mixture (Fig. 2). Amounts of 5
mM and 10 mM of TLCK did not inhibit the conversion of [1,3-3H]DFP into
[l,3-3H]DIP-chymotrypsinderivatives (results not shown).
Specificity of Trypsin and Chymotrypsin Derivatives
The specific inhibition of [1,3-3H]DIP derivatives with TLCK and TPCK
(Figs. 1, 2) allowed us specifically to determine trypsin or chymotrypsin
concentrations in the presence of each other. When 4.2 pg of trypsin and 4.2
pg of chymotrypsin were both incubated in the presence of [1,3-3H]DFP
about 610,000 cpm of [1,3-3H]DIP derivatives were synthesized (Fig. 3A).
When these enzymes were incubated together in the presence of 10 mM
TLCK, only chymotrypsin was labeled (310,000 cpm; Fig. 3B), whereas in the
presence of 10 mM TLCK only chymotrypsin was labeled (320,000 cpm; Fig.
?
-Eo
'5
500
-
400
-
300
-
Trypsinlike and Chymotrypsinlike Enzymes
253
X
2
2
v)
n
200
n
I
Iml 100
-
0
1
0
1
2
3
4
5
6
TRYPSIN (pa)
Fig. 1. Relationship between trypsin concentrations (pglO.1 ml) and [1,3-3H]DIP-trypsin deor presence of 5 mM ( 0 )and 10 mM (0)
of TLCK.
rivatives in the absence of TLCK (0-0)
CHYMOTRYPSIN (pa)
Fig. 2. Relationship between chymotrypsin concentrations (pglO.1 ml) and [1,3-3H]DIP-chymotrypsin derivatives in the absence of TPCK (0-0)or in the presence of 5 mM ( 0 )and
10 m M (0)
TPCK.
3B), and in the presence of 10 mM TPCK only trypsin was labeled (320,000
cpm; Fig. 3C). In the presence of both inhibitors (10 mM TPCK and TLCK)
synthesis of [1,3-3H]DIP derivatives was 95% inhibited (Fig. 3D).
An experiment was done to test whether the specific determination of
trypsin and chymotrypsin in the presence of each other could be done by
using not only pure enzymes but also crude homogenates. Four groups of
female A. aegypti (10 per group) were fed blood on a live chicken. Twenty-
Borovsky and Schlein
254
600
2
400
E
n
0
Y
n
E 200
'
I
'
a
3
Qkl
0
A
B
C
D
A
B
C
D
Fig. 3. Selective synthesis of [1,3-3H]DIP-trypsin and chyrnotrypsin derivatives. Trypsin and
chymotrypsin (4.2 pg each) were incubated with [1,3-3H]DFP and the synthesis of [1,3-3H]DIPtrypsin andlor chyrnotrypsin derivatives was followed without the addition of trypsin and
chyrnotrypsin inhibitors (TLCK and TPCK) (A) in the presence of 10 rnM TLCK (B), in the
presence of 10 rnM TPCK (C), and in the presence of 10 rnM of both TLCK and TPCK (D).
Fig. 4. Selective synthesis of [1,3-3H]DIP-trypsin and chyrnotrypsinlike derivatives in crude
extracts. Female A. aegypti were fed a blood meal and 24 h later rnidguts were removed and
analyzed for (A) [1,3-3H]DIP-chymotrypsinlike derivatives in the presence of 10 rnM TLCK, (B)
[1,3-3H]DIP-trypsinlike derivatives in the presence of 10 mM TPCK, (C) [1,3-3H]DIP-trypsin and
chyrnotrypsinlike derivatives, and (D)inhibition of [1,3-3H]DIP-trypsin and chyrnotrypsinlike
derivatives in the presence of 10 rnM TLCK and 10 mM TPCK.
four hours later, midguts were removed and homogenized in 0.1 ml of 50
mh4 TRIS-HCl buffer, pH 7.9, containing 0.1 M CaC12, and aliquots (equivalent to 0.5 midgut) were assayed for [1,3-3H]DIP derivatives of trypsinlike
and chymotrypsinlike enzymes (Fig. 4C). When 10 mM TLCK (trypsin inhibitor) was added to the reaction mixture, 20% of total [1,3-3H]DIPderivatives
were found to be chymotrypsinlike enzymes (Fig. 4A).On the other hand,
in the presence of 10 mM TPCK (chymotrypsin inhibitor) 77% of the [1,33H]DLP derivatives were trypsinlike (Fig. 4B). When both inhibitors were
added together, approximately 8%of [1,3-3H]DIP derivatives were still synthesized (Fig. 4D), indicating an uncertainty of 8%in the assay.
Time Course of Trypsin Determination in the Mosquito Midgut
During Blood Digestion
Since 77% of the [1,3-3H]DIPderivatives found in the midgut of female A.
aegypti after the blood meal were trypsinlike (Fig. 4B), an attempt was made
to follow the synthesis of trypsinlike enzymes during the blood digestion in
the midgut and to determine quantitatively the amount of trypsin synthesized. Six groups of female A. aegypti (10 per group) were fed blood on a live
chicken, and one group (control) was fed only sugar. At intervals after the
blood meal, midguts were removed and assayed for [1,3-3H]DIP-trypsinlike
derivatives and the amount of trypsin equivalents (nglmidgut) was plotted
against time after the blood meal (Fig. 5). An increase in the amount of
trypsin equivalents was observed in the midgut after the blood meal (Fig. 5 ) .
The number of trypsin equivalents in the midgut of blood fed females
Trypsinlike and Chymotrypsinlike Enzymes
0
10
20
30
40
255
60
U
<
4
HOURS AFTER BLOOD MEAL
(D
Fig. 5. Time course of the amounts of trypsinlike equivalents in the midgut of female A.
aegypti. Groups of females (20 per roup) were fed a blood meal and at intervals midguts
were removed and assayed for [1,3- HIDIP-trypsinlike derivatives which were converted to
trypsin equivalents (ng/ml) by using a calibration curve.
5
reached a peak 24 h after the blood meal (1,400 ng) and thereafter declined
to 1,250 ng at 32 h, reached 150 ng at 48 h, and disappeared at 55 h (Fig. 5).
Quantitative Determination of Trypsinlike Enzymes in Insects and
Leishmania
The reliability of the test to quantitate trypsinlike enzymes in the midgut
of female A . uegypti prompted us to determine the amounts of trypsinlike
enzymes in several insect species. Three groups of insects, insect guts (1-10
per group), or L. major promastigotes (1.2 x lo8 cells per group) were
homogenized or freeze-thawed in 50 mM TRIS-HCl buffer, pH 7.9, containing 0.1 M CaC12. The resulting homogenates or broken cells were centrifuged
in the cold and the supernatants were assayed for [1,3-3H]DIP-trypsinlike
derivatives. Trypsin concentrations were then calculated from the linear
portion of the calibration curve (Fig. 1)and values were expressed as trypsin
equivalents in nglinsect or nglcell (Table 1).Female P. puputusi that were fed
rabbit serum synthesized about 202 ng of trypsin equivalents 30 h after the
serum meal. This amount declined to 37 ng at 48 h. On the other hand,
feeding P. puputusi whole rabbit blood increased the production of trypsin
equivalents by 18% to 235 ng (Table la-c). Rhodnius proZixus or 0. moubutu did
not synthesize trypsinlike enzymes 48 h, 12 days, or 27 days after the blood
meal (Table Id-f). The louse P. humunus synthesized 22 ng of trypsin equivalent by 24 h after the blood meal, but, at 48 h, no trypsin was found (Table
lg,h). The stable fly S. calcifrans synthesized 565 ng of trypsin equivalent 48
h after feeding on a live guinea pig, whereas the house fly M. domestica
synthesized 38% less trypsin equivalents when fed sugar instead of yeast
256
Borovsky and Schlein
TABLE 1. Determination of Trypsin in Several Insect Species and Leishmania*
Insect or parasite
a. P. papatasi, 30 h after a serum meal
b. P. papatasi, 48 h after a serum meal
c. P. papatusi, 30 h after a rabbit blood meal
d. R. prolixus, 48 h after a blood meal
e. R. prolixus, 27 days after a blood meal
f. 0. moubata, 12 days after a blood meal
g. P. humanus, 24 h after a blood meal
h. P. humanus, 48 h after a blood meal
i. S. calcitrans, 48 h after a guinea pig blood meal
j. Pepsin (8 p g )
k. M. domestica, 24 h after a yeast meal
1. M. domestica, 24 h after a sugar meal
m. M. domestica, 48 h after a yeast meal
n. M. domestica, 48 h after a sugar meal
0.L. major promastigotes
p. A . aegypti midgut 24 h after a chicken blood meal
q. Culex nigripalpus midgut 24 h after a chicken blood
meal
r. Culex quinquefasciatusmidgut 24 h after a chicken blood
meal
s . Culic. vuriipennis midgut 24 h after a cow blood meal
Trypsin equivalent
nglinsect f S.E.M.
No. of
insects or
cells (N)
202 f 16
37 & 4
235 f 10
0
0
0
22 f 3
0
565 f 20
0
447 f 30
325 f 20
312 f 24
235 f 16
212 f 12
1,404 f 80
1,302 f 100
30
30
30
3
30
30
30
30
15
15
4.8 x
30
30
1,487 f 110
30
388 & 20
30
-
15
15
15
lo8
*Three groups of insects (1-10 per group) and promastigotes (1.2 x 10' cells per group) were
incubated with [1,3-3H]DFP in the presence of 10 mM TPCK for 18 h at 4°C and analyzed for
[1,3-3H]DIP-trypsinlike derivatives. Trypsin equivalents were calculated from a calibration
curve (Materials and Methods).
(Table lk,l). At 48 h M. domestica fed on yeast synthesized 53% more trypsin
equivalents than flies fed on sugar (Table lm,n). Trypsin equivalents at 48 h
were, however, lower than at 24 h. When pepsin (8 p g ) was tested in the
assay no trypsin equivalents were detected, indicating the specificity of the
determinations (Table l j ) . Leishmania major promastigotes also synthesized
trypsinlike enzymes and about 53 ng of trypsin equivalent was detected per
1.2 x lo8 promastigotes (Table lo). In the midguts of 3 mosquito species
tested 24 h after the blood meal, trypsin equivalents in A . uegypti and Culex
quinquefusciutus were 1,404 ng and 1,487 ng, respectively, whereas Culex
nigripulpus produced 100 ng and 180 ng less, respectively, than did A. uegypti
or Culex quinquefusciutus (Table lp-r). Female Culic. vuriipennis, which take a
much smaller blood meal, synthesized about 388 ng of trypsin equivalent 24
h after the blood meal (Table Is), indicating that the size of the blood meal
determines the amount of trypsin synthesized.
Comparison of [1,3-3H]DIP-TrypsinlikeDerivatives of Mosquitoes and
Culicoides
Borovsky and Schlein [6] reported that several trypsinlike isozymes are
synthesized in the midgut of P. puputusi. Since we already have determined
the trypsin equivalents in the midguts of A. uegypti, Culex nigripulpus, Culex
quinquefusciutus, and Culic. vuriipennis (Table l), we were interested to exam-
Trypsinlike and ChymotrypsinlikeEnzymes
257
ine the isozyme distribution, if any, of the [1,3-3H]DIP-trypsinlike derivatives. Groups of 10 female A. uegypti, Culex nigripulpus, Culex quinquefasciatus,
and Culic vuriipennis were given a blood meal and 24 h later the midguts were
removed, homogenized in 0.1 ml50 mM TRIS-HCl buffer, pH 7.9, containing
0.1 M CaC12, and centrifuged, and the supernatants were incubated in the
cold for 18 h with [1,3-3H]DFP in the presence of 10 mM TPCK. Following
incubation, aliquots (40 pl; equivalent to 4 midguts) of the [1,3-3H]DIPtrypsinlike derivatives were separated on PAGE and analyzed by fluorography (Fig. 6). Culicoides vuriipennis synthesized 2 [1,3-3H]DIP-trypsinlikederivatives, which ran as a slow-moving minor band and a fast-moving major
band (Fig. 6a). Both Culex nigripalpus and Culex quinquefasciatus midguts
synthesized several [1,3-3H]DIP-trypsinlike derivatives that migrated at the
same speed (Fig. 6b,c). Although A . uegypfi (Fig. 6d) and Culex quinquefusciatus midguts synthesized similar quantities of trypsin equivalent (Table l), the
[1,3-3H]DIP-trypsinlike isozymes migrated differently, indicating that there
might be interspecific isozymes differences.
a
b e
d
Fig. 6. PAGE fluorography of [1,3-3H]DIP-trypsinlike isozyrnes. Groups of female A. aegypfi,
Culex nigripalpus, Culex quinquefasciatus, and Culic. variipennis were given a blood meal
and 24 h later rnidguts were removed and [1,3-3H]DIP-trypsinlike isozymes (equivalent to 4
midguts) were separated o n PAGE and analyzed by fluorography (a) Culic. variipennis, (b)
Culex nigripalpus, (c) Culex quinquefasciatus, and (d) A. aegypti.
258
Borovsky and Schlein
DISCUSSION
One of the most important aspects of insect development is the synthesis
of proteolytic enzymes in the midgut in order that food may be digested [2,
6,161. In several blood-sucking insects, most of these enzymes have been
shown to be trypsinlike and chymotrypsinlike [2-6,161. We have developed
a method whereby these enzymes can be monitored by using a trypsin and
chymotrypsin radioactive-labeled inhibitor [1,3-3H]DFPthat covalently binds
these enzymes after phosphorylation of a serine residue at the active site
[6,10,ll]. Using this property, we have shown that the synthesis of [1,33H]DIP-trypsinlike and chymotrypsinlike derivatives is linear with respect to
increase in enzyme concentration (Figs. 1, 2). We have shown, also, that the
assay can detect at least 65 ng of trypsin or chymotrypsin with [1,3-3H]DFP
at a specific activity of 35 Cilmmol. Increase in the specific activity would
allow construction of calibration curves sensitive in the 1-0.1-ng range, which
would bring the technique into the level of sensitivity of detection of many
other biological molecules such as ecdysone and JH [7-91.
The use of 10 mM TPCK and TLCK (chymotrypsin and trypsin inhibitors)
allowed us specifically to quantlfy each enzyme in the presence of the other
(Figs. 3, 4). By means of this technique, we are able to report here for the
first time the amount of trypsinlike enzymes in the midgut of female A.
aegypti during blood digestion. At the peak of synthesis of trypsin, female A .
aegypti midgut contained about 1.4 p g of trypsin equivalent, which decreased
to 1.25 p g at 32 h and disappeared at 55 h (Fig. 5). A similar time-course
determined by enzyme activity has been reported by Graf and Briegel [12]. .
Using our assay we were able to test the amount of trypsin equivalent in
whole animals, midguts, and Leishmania promastigotes (Table 1).In most
insects, the amount of trypsin equivalent per animal was above the lowest
limit of the assay (62 ng; Figs. 1,2). Insects that do not synthesize trypsinlike
enzymes (R. proIixus and 0. moubata), or the enzyme pepsin, served as
controls for the specificity of the assay in that no trypsin equivalent was
found (Table ld-f,j). Sand flies, stable flies, mosquitoes, and biting midges
synthesized trypsinlike enzymes after a blood meal (Table 1).The amount of
trypsin equivalent Synthesized varied with the composition of the blood meal
(serum compared to whole blood Table la,c), which is in accordance with
reports on trypsin activity in female A. aegypti 121. The amount of enzyme
determined is higher at the early phase of blood digestion (24-30 h) and
lower in the later phase (about 48 h, Table la,b,g,h). These results agree with
the reported enzyme activity in mosquitoes and sandflies [5,6,12]. We also
assayed M. domestica, a fly that does not take a blood meal, to find out
whether the amounts of trypsin equivalent were different in flies fed sugar
as compared with those fed on yeast proteins. An increase of 122 ng in
trypsin equivalent was found after a meal on yeast proteins (Table lk,l).
These results are analogous to those in mosquitoes in which the composition
of the blood meal influences proteolytic activity [2]. At 48 h, trypsin equivalent per fly declined, but flies fed yeast proteins contained 77 ng more of
trypsin equivalent than flies fed on sugar (Table lm,n).
Survival of parasites of medical importance in the host midgut depends
on whether they can adapt to the host environment [6,16]. Earlier, we
Trypsinlike and Chymotrypsinlike Enzymes
259
reported that in order to survive, L. major modulated trypsinlike and chymotrypsinlike enzymes in the midgut of P. puputusi [6]. We became interested
to find out whether our assay could detect trypsinlike equivalents in L. major
promastigotes. About 1.2 x 10' promastigotes were needed to detect sufficient trypsinlike equivalents above the 62-ng detection limit, indicating that
the parasites synthesized 1.77 fg of trypsin equivalent per promastigote and
may utilize the enzyme to digest the blood meal for free amino acids.
Comparison between quantities of trypsin equivalents in A. uegypti, Culex
nigripalpus, and Culex quinquefusciutus midguts indicated that, at 24 h, about
1,404-1,487 ng of trypsin equivalent is found in the midgut (Table lp-r).
Since the peak of trypsinlike enzyme synthesis is different in these mosquitoes [5,12], the values reported here are not peak values for the Culex species.
The conclusion from the results reported in Table 1 is that mosquitoes,
sand flies, and other flies have considerable amounts of trypsinlike enzymes
in the midgut above the threshold of 62 ng and that 0.5-0.25 of gut tissue is
needed to detect the enzyme in this assay. Since several isozymes of trypsinlike enzymes that were synthesized in the midgut of P. puputusi and A. uegypti
[6,14] had been successfully tagged with [1,3-3H]DFP, separated on PAGE,
and detected by fluorography, aliquots from the present assays were examined on PAGE by using fluorography. Results indicate that comparisons
between insect species can easily be made by using this technique (Fig. 6).
Although radioimmunoassays are more sensitive than the assay described
here, the drawback of preparing specific antibodies for different enzymes
makes these assays limited and not readily available to many laboratories.
Our assay is general and easy to execute, and it is an attractive method to
use when comparative studies are undertaken of different trypsinlike and
chymotrypsinlike enzymes in insects.
LITERATURE CITED
1. Champlain RA, Fisk FW:The digestive enzymes of the stable fly, Stornoxys culcitruns (L).
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biting fly Stomoxys culcifruns. Arch Insect Biochem Physiol2, 145 (1985).
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culcitruns. Arch Insect Biochem Physiol, 3, 307 (1986).
5. Borovsky D: Proteolytic enzymes and blood digestion in the mosquito, Culex nigripulpus.
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puputusi infected with Leishmania and their possible role in vector competence. Med Vet
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11. Jansen EF, Nutting MDF, Jang R, Balls AK: Inhibition of the proteinase and esterase
activities of trypsin and chymotrypsin by diisopropyl-fluorophosphate:Crystallization of
inhibited chymotrypsin. J Biol Chem, 279, 189 (1949).
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Insect Biochem, 15, 611 (1985).
13. Walsh KA, Wilcox PE: Serine proteases. Methods Enzymol, 19, 31 (1970).
14. Borovsky D: Oostatic hormone inhibits biosynthesis of midgut proteolytic enzymes and
egg development in mosquitoes. Arch Insect Biochem Physiol, 7, 187 (1988).
W.Leammli UK: Cleavage and structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680 (1970).
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