PURIFICATION AND REASSESSMENT OF LIGAND-BINDING BY THE RECOMBINANT, PUTATIVE JUVENILE-HORMONE RECEPTOR OF THE TOBACCO HORNWORM, MANDUCA-SEXTA

The 29 kDa protein from the larval epidermis of the tobacco hornworm, Manduca sexta, that specifically bound photoaffinity analogs of JH I a nd JH II was produced by a recombinant baculovirus (rJP29). The higher of the two molecular weight forms made corresponded to a protein that could be formed by read-through of the TGA termination codon to the f ollowing TAA. The previously reported, apparent high affinity binding of [methyl-H-3]-JH I by rJP29 as measured by the dextran-coated charco al (DCC) assay [Palli et al., Proc Natl Acad Sci USA 91:6191-6195 (199 4)] was found to be artifactual due to endogenous cellular esterases t hat co-purifed with rJP29 through both DEAE cellulose and MonoQ chroma tography. These esterases converted the 10-20 nM labelled JH to JH I a cid and [H-3]-methanol during the 1 h incubation at room temperature. Additionally, DEAE fractions containing rJP29 or from wild-type virus- infected cells were found to bind nonspecifically high amounts of 12, 13-H-3]-JH I acid in the DCC assay. Neither rJP29 nor the cellular est erases had JH esterase activity when assayed on a series of thioether surrogate substrates. When separated from these contaminating esterase s either by hydroxylapatite or affinity chromatography, rJP29 showed l ittle or no detectable binding of [12,13-H-3]-JH I. Yet purified rJP29 bound EBDA, the photoaffinity analog of JH I; and this binding was pr evented by retinol, JH I, methyl farnesoate, methoprene, and xanthophy ll, but not by farnesol and 20-hydroxyecdysone. Therefore, JP29 is not a high affinity JH receptor. (C) 1996 Wiley-Liss, Inc.