CHARACTERIZATION AND LOCALIZATION OF MOSQUITO-GUT RECEPTORS FOR TRYPSIN MODULATING OOSTATIC FACTOR USING A COMPLEMENTARY PEPTIDE AND IMMUNOCYTOCHEMISTRY

The gut receptor of trypsin-modulating oostatic factor (TMOF), a decap eptide hormone that regulates trypsin biosynthesis in the mosquito gut , has been characterized. The binding of TMOF to mosquito gut membrane s reached maximum at pH 7.4 and 24 degrees 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 mi n and 24 degrees C. A synthetic complementary decapeptide NH2-Ile-Leu- Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-COOH (FOMT) for TMOF successfully comp eted with the gut receptor, and specifically bound TMOF (K-d = 4: mu M and K-assoc = 2.5 x 10(5) M(-1)). TMOF binding to gut membranes was c haracterized with FOMT and a specific ELISA to the hormone at 24 and 7 2 h after blood feeding. Two classes of binding sites were found on th e gut membrane; high affinity (K-d1 = 4.6 +/- 0.7 x 10(-7) M; K-assoc = 2.2 x 10(6) M(-1) B-max = 0.1 pmol/gut) and low affinity (K-d2 = 4.4 3 +/- 1 x 10(-6) M; K-assoc = 2.3 x 10(5) M(-1); B-max = 0.2 pmol/gut) . The total binding sites for high and low affinity classes of TMOF pe r put were estimated as 6.3 x 10(10) and 1.1 x 10(11) sites, respectiv ely. Specific binding sites on the put increased after the blood meal and were visualized by immunocytochemical staining. These results sugg est that TMOF regulates trypsin biosynthesis by binding to specific re ceptor sites that are located on the mosquito gut, and that this recep tor can be studied using a complementary peptide approach.