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.
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification / Q000378:metabolism*; D003058:Cockroaches / Q000378:metabolism*; D003979:Diazonium Compounds; D005204:Farnesol / Q000031:analogs & derivatives / Q000378:metabolism; D005260:Female; D006458:Hemolymph / Q000378:metabolism*; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000378:metabolism*; D007700:Kinetics; D009928:Organ Specificity; D010053:Ovary / Q000378:metabolism*
D002352:Carrier Proteins; D003979:Diazonium Compounds; D019476:Insect Proteins; D007605:Juvenile Hormones; C040772:epoxyfarnesyl diazoacetate; C035606:juvenile hormone-binding protein, insect; D005204:Farnesol