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A mosquito-chymotrypsin inhibitor in tissues of adult Aedes aegypti
Comp. Biochera. PhysioL, 1972, Vol. 43B, pp. 137 to 141. PergamonPress. Printed in Great Britain
A M O S Q U I T O - C H Y M O T R Y P S I N I N H I B I T O R IN TISSUES OF A D U L T AEDES A E G Y P T ! Y. J. YANG* and D. M. DAVIES Department of Biology, McMaster University, Hamilton, Canada (Received 24 January 1972)
A b s t r a c t - - 1 . An inhibitor of mosquito and black-fly (simuliid) chymotrypsin, but not of bovine e~-chymotrypsin, was found in the thorax of Aedes aegypti adults. 2. No inhibitor was found in larval mosquitoes or in adult mosquito salivary glands, midgut or abdomen. 3. The inhibitor was activated by incubation at 60°C, not dialysable, and appeared to be a competitive one.
INTRODUCTION INHIBITORS of mammalian proteases do not always inhibit insect proteases. For example, several inhibitors isolated from soybeans and known to inhibit mammalian proteases (Kunitz, 1946) have no effect on the protease activity of flour beetle larvae, Tribolium castaneum (Herbst) (Birk & Applebaum, 1960); while one isolate from the soybeans inhibits insect protease but not mammalian trypsin and chymotrypsin (Birk et al., 1963). Such a specific inhibitor for beetle protease was also found in wheat flour (Applebaum & Konijn, 1966). T h i s report describes the inhibition of mosquito chymotrypsin by tissues of adult Aedes aegypti (L.). MATERIALS AND METHODS Enzyme solutions for chymotrypsinassay were preparedfrom midguts (Yang & Davies, 1968) or excreta (Yang & Davies, 1971b) of larval mosquitoes, A. aegypti, which were maintained as a colonyin this laboratory. The mosquito-chymotrypsininhibitorsolutionwas preparedby homogenizingmosquito tissues, excludingthe midgut, in a glass-tissue grinder. The homogenatewas centrifugedat 3500 rev/minfor I0 rain and the supernatant was frozenat -20°C until needed. Beforethe homogenate was used, it was again centrifuged as above and its concentrationadjusted to 15-20 mosquito tissues per m]. Chymotrypsin activitywas measured with the method described previously (Yang & Davies, 1971a). Inhibitoryactivitywas measured by determiningchymotrypsinactivityin the presence of the tissue homogenate. * Present address: Department of Parasitology, Public Health Laboratory, Toronto, Ontario, Canada. 137
138
Y.J. YANGANDD. M. DAVIES
RESULTS AND DISCUSSION Larval tissue homogenates showed no inhibitory action on mosquito chymotrypsin. However, adult tissue homogenates reduced enzyme activity (Fig. 1). There was little difference in inhibitory activity between male and female tissues or between blood-engorged or sugar-fed females. Inhibition of chymotrypsin by the 1.0
/
o0.8 ~r
o" >: .0.6
o.2
!
0
20
40
REACTION TIME
60
80 MIN
FIG. 1. Effect of adult mosquito tissues on mosquito chymotrypsin activity. ©--C), Chymotrypsin only; 0 - - 0 , chymotrypsin plus the adult tissue homogenate (equivalent to 1.5 mosquito thoraces/assay). tissues may explain the failure of detecting midgut chymotrypsin activity in whole adult mosquito homogenates in other experiments (Yang & Davies 1971a). When the adult midgut alone was used as the enzyme source, chymotrypsin could be detected, whether the fly had fed on blood or not. Tests with homogenates prepared from the adult thorax and abdomen, from which the midgut was removed, revealed inhibitory substance in the thorax, but not in the abdomen (Table 1). Engelmann (1965) detected an inhibitor of roach proteases in the anterior midgut, which may have been produced in the caeca and released into the anterior midgut. We detected no mosquito-chymotrypsin inhibitor in the anterior midgut or salivary glands of the mosquito. It seems likely, therefore, that the inhibitory substance(s) is derived from adult thoracic muscle tissues. When the mosquito chymotrypsin inhibitor was tested against black-fly chymotrypsin (Yang & Davies, unpublished data) and bovine ~-chymotrypsin (3 × -crystallized, Worthington), it only inhibited the former (approximately to the extent of its effect on mosquito chymotrypsin).
MOSQUITO-CHYMOTRYPSIN INHIBITOR IN TISSUES OF AEDES A E G Y P T I
TABLE1--EFFECTOF
139
ADULT MOSQUITO TISSUE HOMOGENATES ON MOSQUITO CHYMOTRYPSIN
Tissue homogenate* Thorax
Percentage inhibition of chymotrypsin
2t 2
28 13 1"5
1
0
1
Abdomen
* Midgut was excluded. t Number of tissues in homogenate per assay. The mosquito tissue homogenate, dialysed against 0.05 M Tris-HCl buffer (pH 7.5) overnight at 4°C, lost no activity. To determine the effect of heat on the inhibitory capacity, dialysed homogenates were incubated at 60°C. At specified times the homogenates were removed and the inhibitory activity was assayed by utilizing mosquito- and/or black-fly-chymotrypsin solution. Incubation of the homogenate at 60°C apparently caused an enhancement of the inhibitory capacity (Fig. 2). However, pre-incubation of the inhibitor with mosquito chymotrypsin solution for periods from 5 to 30 min at 45°C had little effect on its subsequent inhibitory capacity. 40
g ~" 3 0
~
0-•
O
•
hZ
,m, a. 2c
! 20
! 40
I 60
PRE- I N C U B A T I O N T I M E
MIN
FIG. 2. Effect of pre-incubation (the adult tissue homogenate was incubated at 60°C) of adult mosquito tissue on chymotrypsin activity. ©--©, Mosquito chymotrypsin; @--O, black-fly chymotrypsin. The inhibitory capacity of the mosquito tissues seems proportional to the concentration of the inhibitor in the tissues, but an increase of chymotrypsin concentration in a fixed amount of the inhibitor had little effect in raising its
140
Y. J. YaNG aND D. M. DAvms
inhibitory activity. Double reciprocal plots of reaction velocity against the substrate concentration indicated that the adult mosquito tissues contain a competitive inhibitor (Fig. 3). 20
15
~- 10
-1'0
0
1.0
2.0
3-0
4-0
5'0
i/$
FIG. 3. Inhibition of mosquito chymotrypsin by adult mosquito tissues. S, GPNA concentration (mM); V, reaction velocity (O.D. 410 nm). ©--©, Chymotrypsin alone; 0 - - 0 , chymotrypsin plus adult tissue homogenate (equivalent to 1"5 mosquito thoraces/assay).
Acknowledgements--The research was supported by Grant A-130 from the National Research Council of Canada and by a grant from the Ontario Department of University Affairs. Mrs. H. Gy6rk6s prepared the final figures. REFERENCES APPI~EBAUMS. W. & KONIJN A. M. (1966) The presence of a Tribolium-protease inhibitor in wheat. J. Insect Physiol. 12, 665-669. BIRK Y. & APPta~BAUMS. W. (1960) Effect of soybean-trypsin inhibitors on the development and midgut proteolytic activity of Tribolium castaneum larvae. Enzymologia 22, 318-326. BIRK Y., GERTLERA. & KHALEF S. (1963) Separation of a Tribolium-protease inhibitor from soybeans on a calcium phosphate column. Biochim. biophys./tcta 67, 326-328.
MOSQUITO-CHYMOTRYPSININHIBITORIN TISSUESOF A E D E S AEGYPTI
141
ENGELMANN F. (1969) Food-stimulated synthesis of intestinal proteolytie enzymes in the cockroach Leucophaea maderae, j~. Insect Physiol. 15, 217-235. KUNITZM. (1946) Crystalline soybean trypsin inhibitor. ~. gen. Physiol. 29, 149-154. YANGY. J. & DAVIESD. M. (1968) Digestion, emphasizing trypsin activity, in adult simuliids (Diptera) fed blood, blood-sucrose mixtures and sucrose..7. Insect Physiol. 14, 205-222. YANG Y. J. & DAVIES D. M. (1971a) Trypsin and chymotrypsin during metamorphosis in Aedes aegypti and properties of the chymotrypsin. ] . Insect Physiol. 17, 117-131. YANG Y. J. & DAVIES D. M. (1971b) Digestive enzymes in the excreta of ,4edes aegypti larvae, jT. Insect Physiol. 17, 2119-2123.
Key Word Index--Chymotrypsin inhibitor; mosquito chymotrypsin; simuliid chymotrypsin; bovine a-chymotrypsin; Aedes aegypti.
A M O S Q U I T O - C H Y M O T R Y P S I N I N H I B I T O R IN TISSUES OF A D U L T AEDES A E G Y P T ! Y. J. YANG* and D. M. DAVIES Department of Biology, McMaster University, Hamilton, Canada (Received 24 January 1972)
A b s t r a c t - - 1 . An inhibitor of mosquito and black-fly (simuliid) chymotrypsin, but not of bovine e~-chymotrypsin, was found in the thorax of Aedes aegypti adults. 2. No inhibitor was found in larval mosquitoes or in adult mosquito salivary glands, midgut or abdomen. 3. The inhibitor was activated by incubation at 60°C, not dialysable, and appeared to be a competitive one.
INTRODUCTION INHIBITORS of mammalian proteases do not always inhibit insect proteases. For example, several inhibitors isolated from soybeans and known to inhibit mammalian proteases (Kunitz, 1946) have no effect on the protease activity of flour beetle larvae, Tribolium castaneum (Herbst) (Birk & Applebaum, 1960); while one isolate from the soybeans inhibits insect protease but not mammalian trypsin and chymotrypsin (Birk et al., 1963). Such a specific inhibitor for beetle protease was also found in wheat flour (Applebaum & Konijn, 1966). T h i s report describes the inhibition of mosquito chymotrypsin by tissues of adult Aedes aegypti (L.). MATERIALS AND METHODS Enzyme solutions for chymotrypsinassay were preparedfrom midguts (Yang & Davies, 1968) or excreta (Yang & Davies, 1971b) of larval mosquitoes, A. aegypti, which were maintained as a colonyin this laboratory. The mosquito-chymotrypsininhibitorsolutionwas preparedby homogenizingmosquito tissues, excludingthe midgut, in a glass-tissue grinder. The homogenatewas centrifugedat 3500 rev/minfor I0 rain and the supernatant was frozenat -20°C until needed. Beforethe homogenate was used, it was again centrifuged as above and its concentrationadjusted to 15-20 mosquito tissues per m]. Chymotrypsin activitywas measured with the method described previously (Yang & Davies, 1971a). Inhibitoryactivitywas measured by determiningchymotrypsinactivityin the presence of the tissue homogenate. * Present address: Department of Parasitology, Public Health Laboratory, Toronto, Ontario, Canada. 137
138
Y.J. YANGANDD. M. DAVIES
RESULTS AND DISCUSSION Larval tissue homogenates showed no inhibitory action on mosquito chymotrypsin. However, adult tissue homogenates reduced enzyme activity (Fig. 1). There was little difference in inhibitory activity between male and female tissues or between blood-engorged or sugar-fed females. Inhibition of chymotrypsin by the 1.0
/
o0.8 ~r
o" >: .0.6
o.2
!
0
20
40
REACTION TIME
60
80 MIN
FIG. 1. Effect of adult mosquito tissues on mosquito chymotrypsin activity. ©--C), Chymotrypsin only; 0 - - 0 , chymotrypsin plus the adult tissue homogenate (equivalent to 1.5 mosquito thoraces/assay). tissues may explain the failure of detecting midgut chymotrypsin activity in whole adult mosquito homogenates in other experiments (Yang & Davies 1971a). When the adult midgut alone was used as the enzyme source, chymotrypsin could be detected, whether the fly had fed on blood or not. Tests with homogenates prepared from the adult thorax and abdomen, from which the midgut was removed, revealed inhibitory substance in the thorax, but not in the abdomen (Table 1). Engelmann (1965) detected an inhibitor of roach proteases in the anterior midgut, which may have been produced in the caeca and released into the anterior midgut. We detected no mosquito-chymotrypsin inhibitor in the anterior midgut or salivary glands of the mosquito. It seems likely, therefore, that the inhibitory substance(s) is derived from adult thoracic muscle tissues. When the mosquito chymotrypsin inhibitor was tested against black-fly chymotrypsin (Yang & Davies, unpublished data) and bovine ~-chymotrypsin (3 × -crystallized, Worthington), it only inhibited the former (approximately to the extent of its effect on mosquito chymotrypsin).
MOSQUITO-CHYMOTRYPSIN INHIBITOR IN TISSUES OF AEDES A E G Y P T I
TABLE1--EFFECTOF
139
ADULT MOSQUITO TISSUE HOMOGENATES ON MOSQUITO CHYMOTRYPSIN
Tissue homogenate* Thorax
Percentage inhibition of chymotrypsin
2t 2
28 13 1"5
1
0
1
Abdomen
* Midgut was excluded. t Number of tissues in homogenate per assay. The mosquito tissue homogenate, dialysed against 0.05 M Tris-HCl buffer (pH 7.5) overnight at 4°C, lost no activity. To determine the effect of heat on the inhibitory capacity, dialysed homogenates were incubated at 60°C. At specified times the homogenates were removed and the inhibitory activity was assayed by utilizing mosquito- and/or black-fly-chymotrypsin solution. Incubation of the homogenate at 60°C apparently caused an enhancement of the inhibitory capacity (Fig. 2). However, pre-incubation of the inhibitor with mosquito chymotrypsin solution for periods from 5 to 30 min at 45°C had little effect on its subsequent inhibitory capacity. 40
g ~" 3 0
~
0-•
O
•
hZ
,m, a. 2c
! 20
! 40
I 60
PRE- I N C U B A T I O N T I M E
MIN
FIG. 2. Effect of pre-incubation (the adult tissue homogenate was incubated at 60°C) of adult mosquito tissue on chymotrypsin activity. ©--©, Mosquito chymotrypsin; @--O, black-fly chymotrypsin. The inhibitory capacity of the mosquito tissues seems proportional to the concentration of the inhibitor in the tissues, but an increase of chymotrypsin concentration in a fixed amount of the inhibitor had little effect in raising its
140
Y. J. YaNG aND D. M. DAvms
inhibitory activity. Double reciprocal plots of reaction velocity against the substrate concentration indicated that the adult mosquito tissues contain a competitive inhibitor (Fig. 3). 20
15
~- 10
-1'0
0
1.0
2.0
3-0
4-0
5'0
i/$
FIG. 3. Inhibition of mosquito chymotrypsin by adult mosquito tissues. S, GPNA concentration (mM); V, reaction velocity (O.D. 410 nm). ©--©, Chymotrypsin alone; 0 - - 0 , chymotrypsin plus adult tissue homogenate (equivalent to 1"5 mosquito thoraces/assay).
Acknowledgements--The research was supported by Grant A-130 from the National Research Council of Canada and by a grant from the Ontario Department of University Affairs. Mrs. H. Gy6rk6s prepared the final figures. REFERENCES APPI~EBAUMS. W. & KONIJN A. M. (1966) The presence of a Tribolium-protease inhibitor in wheat. J. Insect Physiol. 12, 665-669. BIRK Y. & APPta~BAUMS. W. (1960) Effect of soybean-trypsin inhibitors on the development and midgut proteolytic activity of Tribolium castaneum larvae. Enzymologia 22, 318-326. BIRK Y., GERTLERA. & KHALEF S. (1963) Separation of a Tribolium-protease inhibitor from soybeans on a calcium phosphate column. Biochim. biophys./tcta 67, 326-328.
MOSQUITO-CHYMOTRYPSININHIBITORIN TISSUESOF A E D E S AEGYPTI
141
ENGELMANN F. (1969) Food-stimulated synthesis of intestinal proteolytie enzymes in the cockroach Leucophaea maderae, j~. Insect Physiol. 15, 217-235. KUNITZM. (1946) Crystalline soybean trypsin inhibitor. ~. gen. Physiol. 29, 149-154. YANGY. J. & DAVIESD. M. (1968) Digestion, emphasizing trypsin activity, in adult simuliids (Diptera) fed blood, blood-sucrose mixtures and sucrose..7. Insect Physiol. 14, 205-222. YANG Y. J. & DAVIES D. M. (1971a) Trypsin and chymotrypsin during metamorphosis in Aedes aegypti and properties of the chymotrypsin. ] . Insect Physiol. 17, 117-131. YANG Y. J. & DAVIES D. M. (1971b) Digestive enzymes in the excreta of ,4edes aegypti larvae, jT. Insect Physiol. 17, 2119-2123.
Key Word Index--Chymotrypsin inhibitor; mosquito chymotrypsin; simuliid chymotrypsin; bovine a-chymotrypsin; Aedes aegypti.