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

Abstract

A synthetic analog 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 4th 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 > JH I > JH II > 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 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 2 major proteins in the absence of JH I. The presence of JH I prevented the binding of [3H]EFDA to one protein having a MW of .apprx. 32,000, a MW similar to that of the larval JHBP of Manduca hemolymph.