Purification and reassessment of ligand binding by the recombinant, putative juvenile hormone receptor of the tobacco hornworm, Manduca sexta
1996
Charles, J.P. | Wojtasek, H. | Lentz, A.J. | Thomas, B.A. | Bonning, B.C. | Palli, S.R. | Parker, A.G. | Dorman, G. | Hammock, B.D. | Prestwich, G.D.
The 29 kDa protein from the larval epidermis of the tobacco hornworm, Manduca sexta, that specifically bound photoaffinity analogs of JH I and 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 following TAA. The previously reported, apparent high affinity binding of [methyl-3H]-JH I by rJP29 as measured by the dextran-coated charcoal (DCC) assay [Palli) et al., Proc Natl Acad Sci USA 91:6191-6195 (1994)] was found to be artifactual due to endogenous cellular esterases that co-purified with rJP29 through both DEAE cellulose and MonoQ chromatography. These esterases converted the 10-20 nM labelled JH to JH I acid and [3H]-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 3H]-JH I acid in the DCC assay. Neither rJP29 nor the cellular esterases had JH esterase activity when assayed on a series of thioether surrogate substrates. When separated from these contaminating esterases either by hydroxylapatite or affinity chromatography, rJP29 showed little or no detectable binding of [12,13-3HJ-JH I. Yet purified rJP29 bound EBDA, the photoaffinity analog of JH I; and this binding was prevented by retinol, JH I, methyl farnesoate, methoprene, and xanthophyll, but not by farnesol and 20-hydroxyecdysone. Therefore, JP29 is not a high affinity JH receptor.
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