Assay and identification of juvenile hormone binding proteins in Leucophaea maderae.
We modified a binding assay using polyethylene glycol (PEG) to precipitate bound hormone. Optimum precipitation occurred when reaction mixtures were incubated with 10-40% PEG and 1.25-2.5 mg/ml gamma-globulins for 2-90 min at 4 or 23 degrees C. Results from this assay and from the dextran-coated charcoal assay were similar. Addition of phenylmethylsulfonyl fluoride eliminated nonspecific esterase activity in extracts. JH III-binding macromolecules were identified in hemolymph and ovaries of Leucophaea maderae. These molecules were pronase- and heat-sensitive and saturable. Using Scatchard analysis an average KD of 2.04 (+/- 0.32) X 10(-8) M and 1.91 (+/- 0.80) X 10(-8) M was calculated for hemolymph and ovarian binding proteins. JH III had the highest affinity for binding sites, followed by JH I and JH 0. Various extraction procedures caused changes in JH affinity for both binding proteins. At high concentrations the (+) isomer and mixed isomer preparations of methoprene and hydroprene competed for binding sites. Binding proteins had no affinity for the (-) isomer or for the JH III acid.
D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000032:analysis* / Q000378:metabolism; D011232:Chemical Precipitation; D003058:Cockroaches / Q000032:analysis*; D004950:Esterases / Q000037:antagonists & inhibitors; D005260:Female; D006458:Hemolymph / Q000032:analysis; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000378:metabolism; D010053:Ovary / Q000032:analysis; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011092:Polyethylene Glycols
D002352:Carrier Proteins; D019476:Insect Proteins; D007605:Juvenile Hormones; C035606:juvenile hormone-binding protein, insect; D011092:Polyethylene Glycols; D010664:Phenylmethylsulfonyl Fluoride; D004950:Esterases