Characterizationand localization mosquito-gut receptors of for trypsin modulating oostatic factor using a complementary peptide and immunocytochernistry

DOV

BOROVSKY,*.l C. A. POWELL,t J. K. NAYAR,6 J. EDWIN BLALOCK,1 AND T. K. BAYESS *Institute of Food and Agricultural Sciences, University of Florida, FMEL, Vero Beach Florida 32962, USA; and Agricultural
34954, 35294, USA, USA; Scsences, 1Department Umversity of Physiology of Florida, and Agricultural Research and College Educational Station, Biophysics University University, of Alabama

tlnstitute Pierce,

of Food Florida

Center, at Birmingham
Texas

Fort

Birmingham,

Alabama

5Department

of Entomolog

Texas

A&M

77843, USA

The gut receptor of trypsin-modulating oostatic factor (TMOF), a decapeptide hormone that regulates trypsin biosynthesis in the mosquito gut, has been characterized. The binding of TMOF to mosquito gut membranes reached maximum at pH 7.4 and 24#{176}C. No binding was observed at pH 2.5 and the binding to the membranes declined rapidly at pH 8.0. At equilibrium, maximum binding to the receptor was observed at 60 mm and 24#{176}C. synthetic A complementary decapeptide NH2-lleLeu-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-COOH (FOMT) for TMOF successfully competed with the gut receptor, and specifically bound TMOF (Kd = 4 M and Kassoc = 2.5 x 10 M1). TMOF binding to gut membranes was characterized with and a specific ELISA to the hormone at 24 and 72 h after blood feeding. Two classes of binding sites were found on the gut membrane; high affinity (Kdl = 4.6 0.7 x 10 M; Kassoc = 2.2 X 106 M Bmax = 0.1 pmol/gut) and low affinity (KdI = 4.43 1 x 106 M; Kassoc = 2.3 x 10 M1; Bmax = 0.2 pmollgut). The total binding sites for high and low affinity classes of TMOF per gut were estimated as 6.3 x 1010 and 1.1 x 1011 sites, respectively. Specific binding sites on the gut increased after the blood meal and were visualized by immunocytochemical staining. These results suggest that TMOF regulates trypsin biosynthesis by binding to specific receptor sites that are located on the mosquito gut, and that this receptor can be studied using a complementary peptide approach.Borovsky, D., Powell, C. A., Nayar, J. Blalock, J. E., Hayes, T. K. Characterization and localization of mosquito-gut receptors for trypsin modulating oostatic factor using a complementary peptide and immunocytochemistry. FASEBJ. 8: 350-355; 1994.
ABSTRACT

then

rapidly

declines

and

stops

at

48

TMOF

immediately

after

the

blood

h (4-8). Injecting meal into mosquitos,

proteolytic enzyme

biting midges, flies, and fleas inhibited biosynthesis and blood digestion in the

midgut (5), indicating that TMOF could be used as a diet pill against bloodsucking insects. [3H]TMOF injected into female Aedes aegypti or incubated with gut membranes bound specifically to the
Binding studies was saturable, and was midgut reduced epithelial by increasing

gut. earlier

concentrations

of

the

unlabeled
that the

hormone

(5,

7).
cells

These
have

indicated

TMOF-specific binding sites. Bost et al. (9) and others (for review,
served the binding of peptides from codons by those (termed

see ref 10) have

complementary)

obthat

FOMT

are encoded

by complementary

nucleotide
of hydrophobic of hydrophilic

sequences.
amino amino

This
acids acids

apparently results being complemented and vice versa

(11). Thus, binding of a hormone to a receptor complementary hydrophobic and hydrophiic domains that alter the shapes of the molecules and facilitate binding. Using this concept, many laboratories have demonstrated that many complementary peptides to hormones including corticotropin (ACTH), y-endorphin, or luteinizing hormone-releasing hormone (LHRH) bound the appropriate hormone and that the immune system recognized these may involve
peptides receptor as antigenically similar binding site (10). Thus, to the respective hormone

using
approaches,

complementary

K.,

tide and immunocytochemical sites on isolated gut membranes

TMOF

pepbinding

were identified

and characterized.

MATERIALS Insects

AND

METHODS

Key Words. immunocytochemistry peptide hormone

receptor

binding

ELISA

Larval A. aegypti were reared at 26#{176}C a diet of brewers yeast and lactalon bumin (1:1), under 16:8 h light:dark cycle. Adults were fed on 10% sucrose or on chicken blood. Females were used 3-5 days after emergence. Synthesis of complementary complementary peptide to TMOF

(FOMT)

ANAUTOGENOUS

FEMALE

MOSQUITOES

LACK

reserve

proteins

and take a blood meal in order to develop main digestive enzyme in the midgut of these sin (1-3). After the blood meal, the midgut start to synthesize midgut (3). At highest level, the trypsin follicular and

their

eggs.

The

A peptide

(NH2-Ile-Leu-Gly-Arg-Gly-Gly-Glyin the laboratories ofT K. Hayes at

females is trypepithelial cells is into at the the

Gly-Gly-Gly-COOH)

was synthesized

24 h, when release
of TMOF

the enzyme is secreted trypsin biosynthesis of the then ovary the

To whom

correspondence

should

be addressed,

at: University

of

epithelium

synthesize
mum declines and the

and

TMOF

into

the hemolymph; binds

to the gut biosynthesis,

synthesis epithelial

is at 36 h and to stop trypsin

begins to maxisynthesis

Florida-IFAS, Florida Medical Entomology Laboratory, Vero Beach, FL 32962, USA. 2Abbreviations: ACTH, corticotropin; LHRH, luteinizing hormone-releasing hormone; TMOF, trypsin-modulating oostatic

stops

at 48 h. TMOF cells

and

sig-

nals

which

factor; TSTB, Tris saline plementary peptide.

Triton

buffer;

FOMT,

TMOF

com-

350

0892-6638/94/0008-0350/$0l

.50. FASEB

RESEARCH COMMUNICATIONS

z
0
U-

0 2

I-

TIME

(mm)

Figure 1. Time course of specific binding of TMOF to gut membranes. Each incubation well was incubated at 24#{176}C contained and gut membranes from seven female A. aegypti that were fed blood 24 h earlier and TMOF (2.0 tg). Nonspecific binding (17-26% of total binding) was subtracted from each point and was determined by incubating FOMT (8.0 tg) in parallel wells. Each point is the average of two determinations.

pH Figure
3. Relationship between pH and binding of TMOF to gut membranes. Each well contained gut membranes from seven female A. aegypti and TMOF (2.0 tg) and was incubated for 1 h at 24#{176}C 20 mM Glycine-HCI in buffer for pH 2.6, 20 mM Tris maleate buffer for pH 4.0 to 6.5, and 20 mM Tris HCI buffer for pH 7.0 to 8.5. Each point represents an average of two determinations.

Texas A&M University and J. E. Blalock, University of Alabama at Birmingham. The peptide purity and homogeneity was confirmed by reversed phase C18 HPLC and by fast atom bombardment mass spectrometry in which the peptide exhibited an abundant ion at m/z 799.7.

7.5) in such a way that 1 l of membrane preparation was equivalent gut. Only freshly prepared gut membranes were used for the binding ments.

to one

experi-

Preparation

of gut membranes

Binding

of TMOF

to gut membranes

Guts were dissected from female A. aegypti 24 and 72 h after the blood meal. The guts (100 per group) were homogenized in 0.1 ml 20 mM Tris-maleic acid buffer, pH 6.5, containing 500 mM NaC1, 1 mM EDTA, 10 ig bacitracm, 10 sg aprotonin, 50 sg BSA, and 1 mM PMSF at 5#{176}C a Teflon with homogenizer. The homogenate was then centrifuged at 14,000 x g at 5#{176}C and the supernatant was removed. The pellet was rehomogenized, sonicated for 60 s, and recentrifuged for 10 mm at 5#{176}C. The pellet was then resuspended at 5#{176}C0.1 ml TBS (20 mM Tris-HCI, 500 mM NaCl, pH in

Binding assays were carried Out in Costars nitrocellulose filter strip system (Cambridge, Mass.). Membranes in wells were wetted with 100 sl TBS, pH 7.5, for 5 mm, and the buffer was removed from the wells by gentle vacuum. Gut membranes (5-10 per well) or different amounts of TMOF complementary peptide (FOMT) were added and allowed to incubate for 30 mm in

1.2

0.8 E 0.6

TBS (100 tl) at room temperature. The solution was then removed under gentle vacuum, and the wells were washed three times with TBS. The wells were blocked with TBS, 1% Tween 20 (Sigma, St. Louis, Mo.), and 2% BSA (Sigma), pH 7.5 (100 il), for 30 mm at room temperature. The blocking solution was removed from the wells by vacuum and different amounts of TMOF were added and allowed to incubate in the wells for 1 h at room temperature. To determine nonspecific binding, TMOF complementary peptide (FOMT) was added at concentrations fourfold higher than TMOF. After incubations, the solutions were removed from the wells with a gentle vacuum and the membranes were washed three times with TBS, 0.05% Tween 20, pH 7.5, and 1% rabbit anti-TMOF serum (100 s1) (in TBS, pH 7.5) was added and the filter strips were incubated at 4#{176}C 18 h. Strips for were then developed with goat anti-rabbit immunoglobulin covalently linked with alkaline phosphatase and p-nitrophenyl phosphate (12). The absorbances of control wells containing gut membranes were subtracted from wells that contained TMOF and gut receptor. Each assay was repeated three
times.

0.4

Immunocytochemistry

of TMOF

binding

to mosquito

gut

0.2

0 0 10 20 30 FOMT (jg) 40 50

Figure 2. Effect of FOMT on the binding of TMOF to gut membranes. Gut membranes from seven female mosquitoes were incubated at 24#{176}C I h with TMOF for (2.0 tg) and increasing amounts of FOMT (0-40 ig). Each point represents an average of two determinations.

Midguts of sugar-fed and blood-fed female A. aegypti were dissected in phosphate-saline buffer and fixed in Bouin Hollandes sublimate fixative for 24 h (13). After fixation, the tissues were washed in distilled water for 12 h, dehydrated in 75%, 95%, and 100% ethanol, treated with xylollethanol, and embedded in Paraplast. Serial sections (7 sm) of midguts were cut using a rotary microtome and attached to glass slides with egg albumin. Tissue sections were immunochemically stained using a modified technique described by Vandesande (14). Briefly, the tissue sections were deparaffinated and hydrated in 100% xylol (two changes of 5 mm), 100% ethanol (three changes of 3 mm), and 1 mm in distilled water. The sublimate was removed by incubating tissue sections in Lugol (two times, for 2 mm) and in 5% sodium
thiosulfate. The tissue sections were washed with distilled water and

equilibrated

for 5 mm in Tns saline Triton buffer (TSTB) containing 10 mM Tris-HCI, 150 mM NaCI, 1 mM NaN3, pH 7.6, 0.1% Triton X-lOO. Tissue sections were then incubated for 45 mm with preimmune goat serum

MOSQUITO

TMOF GUT RECEPTOR

351

RESEARCH COMMUNICATIONS

1000

2000

3000
S

4000

5000

1500

TMOF (hg)

5000 Binding of TMOF to its complementary peptide TMOF (FOMT). Increasing amounts of TMOF (0-2,800 ng) were incubated with FOMT bound to nitrocellulose wells for 1 h at 24#{176}C. Figure 6. Specific binding of TMOF to gut membranes of female Scatchard analysis of the data is shown in the inset. The data are A. aegyptithat were fed blood 72 h earlier. Each well contained seven a typical experiment of two independent determinations. A dissocigut membranes and different amounts of TMOF (0-4000 ng). ation constant of 4 x 10-6 M was calculated from these data. Other conditions are as in Fig. 4. The data are a typical experiment of three independent determinations. High (Kdl = 3.6 x 10 M) and low (Kd2 = 6.2 x 10-6 M) dissociations constants were calcu(Sigma, St. Louis, Mo.), diluted 1:5 in TSTB, and rinsed several times in lated from these data. TSTB to remove unbound proteins. Each slide with tissue sections was then incubated with TMOF (0.2 ml, 130 sg) in TSTB for 1 h, washed in TSTB, and incubated overnight at room temperature with rabbit antiserum against TMOF (1:300 dilution in TSTB). After incubation, slides with tissue secRESULTS AND DISCUSSION tions were washed, incubated for 25 mm with goat anti-rabbit antibody (Sigma), diluted 1:30 in TSTB, washed and incubated for 25 mm with Preliminary data have indicated that mosquito gut has a peroxidase-antigoat-perodixase-complex diluted 1:300 in TSTB, washed in TSTB, and developed in 3-3-diaminobenzidine. The tissue sections were saturable receptor for TMOF (5, 7). To characterize the then dehydrated in increasing concentrations of ethanol and clarified in TMOF receptors, gut membranes were isolated 24 h after 100% xylol and mounted with Permount. 4.

Figure

2000

3000 TMOF (ng)

Figure

5. Specific

binding

of TMOF

to gut membranes

of female

A. aegypti that were fed blood 24 h earlier. Each well contained seven gut membranes and various amounts of TMOF (0-5,000 ng). Nonspecific binding was determined by incubating TMOF in the presence of FOMT and represented about 26% of total binding at
the maximum. Scatchard analysis of the data is shown in the inset. The data are a typical experiment of three independent determinations. High (Kdl 5.7 x 10 M) and low (Kd2 = 2.2 x 10-6 M) dissociation constants were calculated from these data.

the blood meal from seven female A. aegypti, homogenized, sonicated, and bound to nitrocellulose membranes in nitrocellulose strips (7 gut membranes per well) and incubated for different periods (0-180 mm) with TMOF in the presence and absence of its complementary peptide (FOMT). The specific binding of TMOF (2.0 g) to the gut membranes reached an equiibrium after incubation for 60 mm at room temperature (Fig. 1), and all subsequent incubations were carried out at 60 mm and room temperature. The binding of TMOF (2.0 &g) to the gut membranes was reversed in the presence of FOMT Maximum displacement of TMOF from the gut membrane by FOMT was achieved with amounts twofold higher than TMOF; increasing the amount of FOMT up to 40 eg did not displace more of the hormone (Fig. 2). Thus, about 26% of the binding of the hormone to the gut membrane is nonspecific. No inhibition of TMOF binding to gut membranes was observed when renm inhibitor (NH2Pro-His-Pro-Phe-His-Phe-Phe-Val-TyrLys-COOH) (4 ftg) was substituted with FOMT (results not shown) indicating that the binding of TMOF to FMOT is specific. Incubation of the hormone at different pHs indicated that optimum binding was between pH of 5.5 and 7.4, with maximum binding at pH 7.4 (Fig. 3). As the pH values for the hemolymph of three mosquito species Aedes taeniorhynchus, Culexpipiens, and Aedes dorsalis are between 7.5 and 7.6 (15), pH
7.4 was used

to

characterize

the

binding

of

the hormone to

its receptor.

To determine the affinity of TMOF for its complementary peptide, nitrocellulose wells were incubated with FOMT (7 eg), and the wells were washed to remove unbound peptide, blocked, and incubated with increasing concentrations of
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RESEARCH COMMUNICATIONS
acids and peptides released from the blood meal make quantitation difficult, e.g., a membrane preparation of sugar-fed female has I ig protein per gut, whereas a membrane preparation at 24 h after blood digestion has 4 ig protein per gut. When gut membranes were prepared 24 h and 72 h after the blood meal and analyzed for specific binding of TMOF by the method of Scatchard (16), two classes of binding sites were found (Fig. 5 and Fig. 6). The apparent high affinity constants (Kdj) for membrane preparations at 24 and 72 h were similar (5.7 x 10 M and 3.6 x 10 M, respectively). The apparent low affinity constants (Kd2) for membrane preparations at 24 and 72 h were 2.2 x 106 M and 6.2 x 10-6 M, respectively (Fig. 5 and Fig. 6). Because the binding constants at 24 h and 72 h were similar, we expressed the results as the average SEM (n = 6) of three determinations at 24 h and three determinations at 72 h. The apparent average high-affinity dissociation constant (Kdl) is 4.6 0.7 x 10 M and the association constant (Kassec) is on the order of 2.2 x 106 M, which is 10-fold higher than for the complementary peptide, FOMT (Fig. 4). The maximum amount of TMOF bound per gut membrane preparation (Bmax) to the high affinity receptor is 0.1 pmol/gut which is equivalent to 6.3 x 1010 binding sites per gut. The average low affinity dissociation and association constants were 10-fold lower (Kd2 4.43 I x 10-6 M and Kassoc = 2.3 x 10 M-1). The maximum amount of TMOF bound per low affinity sites (Bmas) was calculated as 0.2 pmol/gut, which is the equivalent of 1.1 x 10 binding sites. Because the total amount of TMOF in one pair of ovaries of a female mosquito is about 112 pmol or 117 ng (12), there is a sufficient amount of hormone in one female to bind both the high and low affinity sites when the biosynthesis of trypsin is rapidly terminated 24 to 48 h after the blood meal. High and low affinity binding sites of insect hormones have been reported for the juvenile hormone receptors of the cockroach Leucophaea maderae and for the ecdysteroid receptors of female Drosophila melanogaster. A 10-fold difference between the high and low affinity constants was observed (18, 19). A molecular model of the complementary peptide superimposed onto TMOF was developed using SYBYL molecular modeling software version 5.2 (Tripos Associates Inc., St. Louis, Mo.). The similitaries between the TMOF low affinity binding constants for the gut receptors and the TMOF binding constants for FOMT suggested that a comparison of the two peptides might yield additional structural relationships. Molecular models for the two peptides were initially constructed independent of one another. Simple energy minimizations for each peptide resulted in a helical strucutre for the COOH-terminal polyammno acid tail of each compound. NMR studies have recently confirmed that the range of solution conformations for TMOF are dominated by this feature (5, 6, 20). Use of the Fit command in SYBYL aligns the superimposed structures to display the similarities (Fig. 7). The negative charge of the Asp2 side chain on TMOF and the positive charge of the Arg4 side chain on FOMT are in similar positions in the two structures. That predicts one logical site among many for the two peptides to interact. However, computer modeling alone cannot prove the conformation of TMOF in the gut receptor or where is is bound to FOMT. However, the prediction of
such might with tainly similar structures account for some its complementary can be used by simple computational chemistry aspects of the interaction of TMOF peptide, and that prediction cera structural hypothesis to plan

stereoview of TMOF and its complementary peptide FOMT represented by a stick model. TMOF hydrophilic amino acids Tyr and Asp2 are colored red (bottom), whereas the hydrophobic amino acids (Pro3, Ala4, Pro5#{176}) are colored green. The NH2 terminus of both peptides is at the bottom, the COOH terminus is on top. FOMT backbone atoms are: white carbons, red oxygens, and blue nitrogens.

Figure 7. A superimposed

TMOF

h), each

equilibrium had been reached (1 for binding of TMOF using ELISA (12). Analysis of the binding data using Scatchard analysis (16) confirmed that the binding was linear with a single class of binding sites and an apparent dissociation constant (Kd) of 4 x 10-6 M and an affinity (Kassoc) of 2.5 x 10 M1 (Fig. 4). A single class of binding sites is expected from complementary peptide studies (9) with a theoretical binding ratio of 1:1 if all the FOMT (7 peg) bound each well and if each TMOF molecule binds one FOMT molecule. Maximum binding (Bmae) that was calculated from the Scatchard analysis (Fig. 4) indicated that 5300 ng of TMOF bound 7000 ng of FOMT (1:0.76 binding ratio) if 100% of FOMT bound each well. If, on the other hand, only 76% of FOMT bound each well then the ratio of binding of TMOF to FOMT is 1:1, suggesting a monomolecular interaction. Complementary peptides to ribonuclease S peptide (20 amino acids) and substance P (9 amino acids) also exhibited similar dissociation constants (1.2 x 10-6 M and 6 x 10-6 M, respectively) (10). The ability of FOMT to compete specifically with the binding of the hormone to its gut receptor enabled us to characterize the binding of TMOF to the gut receptor. Important physiological changes in the female mosquito occur after the blood meal; trypsin is synthesized in the midgut, and the egg yolk protein vitellogenin is synthesized by the fat body (3, 17). To characterize the binding of TMOF to its receptor, midgut membranes were removed from female mosquitoes 24 and 72 h after the blood meal and assayed for specific TMOF binding. Because binding reached equilibrium in 60 mm at room temperature with seven gut membrane equivalents (Fig. 1), all incubations were followed for 60 mm using membranes from seven guts. The results are expressed as specific binding per gut rather than per amount of protein, because after the blood meal free amino

(0-2.8

tg).

After

well was analyzed

to initiate

investigations of the TMOF-FOMT complex. Similar comparative computational models have been used to predict effective inhibitors of cysteine and serine proteases from the

MOSQUITO

TMOF

GUT

RECEPTOR

353

RESEARCH COMMUNICATIONS

Figure 8. Immunolocalization of TMOF binding to mosquito midgut receptor 72 h after the tm) were incubated with TMOF and polyclonal antibodies. Distinct binding of TMOF to its of the transverse section (b, binding area stained dark between arrows). Sections a and c on either did not show any binding. d) fourfold magnification of the binding region in b, e) Gut incubated magnification of negative region between arrows in e. He, hemolymph side, outside gut, Lu,

blood meal. Serial transverse sections (7 receptor was observed on the small area side of the section b with TMOF receptor without TMOF, no binding, f) fourfold lumen side, inside gut.

infectious larvae for the disease schistosomaisis (21). The specificity of TMOF binding to the mosquito gut was also investigated using an immunocytochemical approach. Midguts were removed from female A. aegypti before and after the blood meal, and the guts were sectioned, fixed, and incubated with TMOF, and assayed for binding using TMOF specific polyclonal antibodies (12). Three controls were used: 1) tissue was incubated without TMOF, 2) anti-

bodies were preabsorbed with TMOF before the tissue was incubated with TMOF, and 3) TMOF and FOMT were preincubated for 1 h and then reincubated with the tissue. Positive immunocytochemical staining was detected only in the presence of TMOF (Fig. 8). Distinct regions were located on the gut to which TMOF bound (Fig. 8b, d). On the other hand, these regions were absent when TMOF was not present (Fig. 8e, f) or if the polyclonal antibodies serum or

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BOROVSKY

Et AL.

RESEARCH COMMUNICATIONS
FOMT was preincubated with TMOF (results not shown). The specificity of the binding to certain regions on the mosquito gut was demonstrated when three consecutive gut sections (7 jm in thickness) were cut, fixed, and incubated with TMOF. Only the middle gut section bound Ti OF (Fig. 8b) whereas the other sections apparently did not have TMOF receptors and no staining was observed (Fig. 8a, c). Similar results were observed when guts were removed 24 and 48 h after the blood meal and analyzed by immunocytochemistry (results not shown). When whole mosquitoes were sectioned longitudinally and the sections were incubated with TMOF and stained using the immunocytochemical method, the hormone bound only to the midgut and to the follicular epithelium 48 h and 72 h after the blood meal. Previous studies had shown that the follicular epithelium synthesizes and stores the hormone (8). Thus, we conclude that TMOF specific binding sites are located on the
mosquito receptor, of trypsin This visiting midgut, and upon binding of the hormone the to its a signal is transmitted biosynthesis in the that initiates midgut. termination 13. 14. 7. Borovsky, D., Carlson, D. A., Griffin, P. R., Shabanowitz, J., and Hunt, D. F. (1993) Sequencing and characterization of trypsin modulating oostatic factor. In Host Regulated Developmental Mechanisms in Vector Arihropods, Proceedings of the Third Symposium, Vero Beach, Florida (Borovsky, D., and Spielman, A., eds) pp. 36-47, University of Florida-IFAS, Florida Medical Entomology Laboratory, Vero Beach 8. Borovsky, D., Song, Q., Ma, M., and Carlson, D. A. (1993) Bio8ynthesis, secretion and immunocytochemistry of trypsin modulating factor of Aedes aegypti. Arch. Insect Biochem. Physiol. (In press) 9. Bost, K. L., Smith, E. M., and Blalock, J. E. (1985) Similarity oostatic between

10.

11.

12.

work was partially supported by the Lady Davis Foundation professorship award to D. B., by FDACS contract 1415 to E. B. This Station

D. B., and by PHS grants DK 38024 and NS 29719 toJ. paper is part of University of Florida-IFAS Experimental Journal Series No. R-03448.

15. 16. 17.

REFERENCES
1. Gooding, R. H. (1966) In vitro properties of proteinases in the midgut of adult Aedes aegypti (Linaeus) and Culexfatigans (Weidmann). Comp. Biochem. PhysioL 17, 115-127 2. Briegel, H., and Lea, 0. A. (1975) Relationship between protein and proteolytic activity in the midgut of mosquitoes. j Insect Physiol. 21, 1597-1604 3. Borovsky, D., and Schlein, Y. (1988) Quantitative determination of trypsinlike and chymotrypsinlike enzymes in insects. Arch. Insect Biochem. Physiol. 8, 249-260 4. Borovsky, D. (1985) Isolation and characterization of highly purified mosquito oostatic hormone. Arch. Insect Biochens. Physzol. 2, 333-349 5. Borovsky, D., Carlson, D. A., Griffin, P. R., Shabanowitz, J., and Hunt, D. F. (1990) Mosquito oostatic factor: a novel decapeptide modulating trypsin-like enzyme biosynthesis in the midgut. FASEBJ 4, 3015-3020 6. Borovsky, D., Carlson, D. A., Griffin, P. R., Shabanowitz, J., and Hunt, D. F. (1993) Mass spectrometry and characterization of Aedes aegypti trypsin modulating Oostatic factor (TMOF) and its analogs. Insect Bioche,n. Molec. Biol. 23, 703-712

18.

19.
20.

21.

the corticotropin (ACTH) receptor and a peptide encoded by an RNA that is complementary to ACTH mRNA. Proc. Nail. Aca#{128} USA 82, Sd. 1372-1375 Jarpe, M. A., and Blalock, J. E. (1993) Complementary peptides: applications of the molecular recognition theory to peptide and protein purification and design. In Peptides: Design, Synthesis, and Biological Activity (Busava, C., and Ananthramaiah, G. M., eds) Birkh#{228}user, Boston (In press) Blalock, J. E., and Smith, E. M. (1984) Hydropathic anticomplementarily of amino acids based on the genetic code. Biochem. Blophys. Res. Commun. 121, 203-207 Borovsky, D., Powell, C. A., and Carlson, D. A. (1992) Development of specific RIA and ELISA to study trypsin modulating oostatic factor in mosquitoes. Arch. Insect Biochem. Physiof 21, 13-21 Sternberger, L. A. (1979) Immunocytochemistry. Wiley Medical Publication, New York Vandesand; P. (1983) Immunocytochemical double staining techniques. In Immunocytochemistry (Cuello, A. C., ed) pp. 257-272, Wiley, New York Clements, A. N. (1992) The Biology of Mosquitoes. Chapman & Hall, London, 509 pp. Scatchard, G. (1949) The attractions of proteins for small molecules and ions. Ann. N Y Acad. Sci. 31, 660-672 Borovsky, D., Thomas, B. R., Carlson, D. A., Whisenton, L. R., and Fuchs, M. S. (1985) Juvenile hormone and 20-hydroxyecdysone as primary and secondary stimuli of viteilogenesis in Aedes aegypti. Arch. Insect Biochem. Physiol. 2, 75-90 Engelmann, F., Mala, J., and Tobe, S. S. (1987) Cytosolic and nuclear receptors for juvenile hormone in fat bodies of Leucophaea maderae. Insect Biochcn. 17, 1045-1052 Handler, A. M., and Maroy, P. (1989) Ecdysteroid receptors in Drosophila melanogaster adult females. Mol. Cell. Endocrinol. 63, 103-109 Curto, E. V., Jarpe, M. A., Blalock, J. E., Borovsky, D., and Krishna, N. R. (1993) Solution structure of trypsin modulating oostatic factor is left-handed helix. Biochem. Biophys. Res. Commun. 193, 688-693 Ring, C. S., Sun, E., McKerrow, J. H., Lee, C. K., Rosenthal, P. J., Kuntz, I. D., and Cohen, F. E. (1993) Structure-based inhibitor design by using protein models for the development of antiparasitic agents. Proc. NaIl. Acad. Sci. USA 90, 3583-3587 Received for publication October 14, 1993. Accepted for publication November 30, 1993

ERRATUM
The authors wish to correct a paragraph that appeared in a research communications article on page 115 in the January 1994 issue of The FASEB Journal [Chinsky, J. M., Bohlen, L. M., and Costeas, P. A. (1994) Noncoordinated responses of branched-chain a-ketoacid dehydrogenase subunit genes to dietary protein. FASEB j 8, 114-120]. Changes are in boldface.

protein, 10.0% fat, and 81.70% carbohydrate; and 50% ProteinPurified Diet (#5786C) containing 50.5% protein, 13.50% fat, and 23.35% carbohydrate. The latter two diets are isocaloric, providing 4.17 Kcal/g of calculated digestible energy (physiologic fuel value). Standard laboratory rodent diet has a potential energy content (gross energy) of 4.0 Kcal/g but provides only 3.3 Kcal/g according to its calculated physiologic fuel value, as determined by the manufacturer. All three diets contain similar mineral and fiber content. The mice were fed these diets and water on an
ad libitum basis. In general, no significant differences in the volumes of food or water consumed were noted in the groups of mice fed these three different isocaloric diets. Experimental groups, containing at least four mice per group, were age-matched, sex-matched, and when possible, litter-matched.

METHODS
Animals All mice [C575L/6J, ob/ob and lean (oh/i., +1+) littermatesl were obtained from the Jackson Laboratory, Bar Harbor, Me. The diets used were obtained from Purina Tests Diets, Richmond, Ind., and included standard Laboratory Rodent Diet (#5001) containing 23.4% protein, 5.5% fat, and 49.0% carbohydrate; Protein Free Purified Diet (#5765C) containing 0%

All experiments were repeated at least twice with different groups of mice. Only female mice were used because their weight gain during this age (6.5-8.5 weeks) is minimal compared to male mice and standard weight gain curves available from the Jackson Laboratories. Results are shown for those experimental groups of mice who consumed equivalent volumes of food and as a group demonstrated similar weight changes during the experimental periods (1-2 weeks). The weight changes observed in C57BL/6J mice on standard laboratory chow (23% protein) during the experimental periods (6.5-8.5 weeks of age) averaged 3.97% ( 2.25 for 3 experiments involving n = 12 mice) per week, similar to those reported by the supplier, the Jackson Laboratories.3

MOSQUITO TMOF GUT

RECEPTOR

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