Quantitative determination of trypsinlike and chymotrypsinlike enzymes in insects.код для вставкиСкачать
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  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  and Borovsky  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  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 . . 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 . 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 . 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). Ohio J Sci, 56, 52 (1956). 2. 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