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 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 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 min and 24 degrees C. A synthetic complementary decapeptide NH2-Ile-Leu-Gly-Arg-Gly-Gly-Gly-Gly-Gly-Gly-COOH (FOMT) for TMOF successfully competed with the gut receptor, and specifically bound TMOF (Kd = 4 microM and Kassoc = 2.5 x 10(5) M-1). TMOF binding to gut membranes was characterized with FOMT 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 (Kd1 = 4.6 +/- 0.7 x 10(-7) M; Kassoc = 2.2 x 10(6) M-1 Bmax = 0.1 pmol/gut) and low affinity (Kd2 = 4.43 +/- 1 x 10(-6) M; Kassoc = 2.3 x 10(5) M-1; Bmax = 0.2 pmol/gut). The total binding sites for high and low affinity classes of TMOF per gut were estimated as 6.3 x 10(10) and 1.1 x 10(11) 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
D
Institute of Food and Agricultural Sciences, University of Florida, FMEL, Vero Beach 32962.
Powell
C A
CA
Nayar
J K
JK
Blalock
J E
JE
Hayes
T K
TK
D000595:Amino Acid Sequence; D000818:Animals; D009033:Culicidae / Q000737:chemistry*; D005260:Female; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000378:metabolism*; D018000:Receptors, Peptide / Q000032:analysis* / Q000502:physiology
D009842:Oligopeptides; D018000:Receptors, Peptide; C086560:trypsin-modulating oostatic factor receptor; C065612:Peptide oostatic hormone