Photoaffinity labeling of the hemolymph juvenile hormone binding protein of Manduca sexta.

A synthetic analogue of the insect juvenile hormone (JH) III, 10,11-epoxy[10-3H]farnesyl diazoacetate [( 3H]-EFDA), binds to several proteins in a partially purified preparation of hemolymph protein from fourth instar larvae of Manduca sexta when irradiated with UV light. Approximately 80% of this binding could be inhibited by the addition of excess unlabeled JH I. To compare the relative affinity of EFDA for the juvenile hormone binding protein (JHBP) with that of the various JH homologues, the ability of unlabeled EFDA and JH homologues to displace [3H]JH I from binding sites was measured. The relative affinities were EFDA greater than JH I greater than JH II greater than JH III. When Scatchard analysis of the binding of [3H]EFDA or [3H]JH I to the larval JHBP was performed, an estimated apparent KD of 4.5 X 10(-8)M was found for EFDA, whereas for JH I a slightly higher KD of 8.8 X 10(-8) M was calculated. To determine if [3H]EFDA bound at the JH I binding site, displacement of [3H]JH I from the JHBP complex with unlabeled JH I, JH II, and JH III was compared to the displacement of [3H]EFDA with the same homologues. The results demonstrated that the photoaffinity label bound covalently at the JH I binding site on the hemolymph binding protein of Manduca sexta. Fluorescence autoradiography of [3H]EFDA photoaffinity labeled proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that [3H]EFDA bound covalently to two major proteins in the absence of JH I.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific photoaffinity label for hemolymph and ovarian juvenile hormone-binding proteins in Leucophaea maderae.

A tritium-labeled diazocarbonyl juvenile hormone (JH) analog, (10-[10,11-3H]epoxyfarnesyl diazoacetate, [3H]EFDA), covalently bound to proteins in both hemolymph and ovarian extracts when reaction mixtures were irradiated with UV light. The addition of various concentrations of unlabeled JH III selectively inhibited [3H]EFDA photoattachment to proteins. Using the Scatchard method of analysis, [3H]EFDA bound specifically and with relatively high affinity (KD = 1.5 X 10(-6) M) to a macromolecule in each extract, although nonspecific binding to other molecules was also present (20-50%). To determine if [3H]EFDA bound at the JH III-binding site on the binding proteins, radioactive [3H]JH III or [3H]EFDA was complexed with proteins in the presence of various concentrations of either unlabeled JH III or JH I under equilibrium conditions. The results demonstrated that the natural hormone, JH III, displaced both bound labeled ligands 4.1 +/- 0.5 times better than the homolog JH I. Thus, the photoaffinity label [3H]EFDA bound at the same site on the protein as [3H] JH III. Fluorescent autoradiography of [3H]EFDA-labeled proteins separated by sodium dodecyl sulfate electrophoresis revealed that several proteins in both hemolymph and ovarian extracts bound [3H]EFDA. To determine the specificity of binding, extracts were irradiated with UV light in the presence of unlabeled JH III and [3H]EFDA. The results demonstrated that JH III prevented photoattachment of [3H]EFDA to a major protein in each extract. The molecular weight of these proteins was estimated at approximately 200,000 for both the hemolymph protein and the ovarian protein.

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.