Purification and Reassessment of Ligand Binding by the Recombinant, Putative Juvenile Hormone Receptor of the Tobacco Hornworm, Manduca sexta

Jean Philippe Charles, Hubert Wojtasek, Anthony J. Lentz, Beth Ann Thomas, Bryony C. Bonning, Subba Reddy Palli, Anthony G. Parker, György Dorman, Bruce D. Hammock, Glenn D. Prestwich, Lynn M. Riddiford

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28 Scopus citations

Abstract

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 [12,13-3H]-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.

Original languageEnglish (US)
Pages (from-to)371-393
Number of pages23
JournalArchives of Insect Biochemistry and Physiology
Volume31
Issue number4
StatePublished - 1996

Keywords

  • Cellular esterases
  • JH esterase
  • Juvenile hormone receptor
  • Manduca sexta
  • Photoaffinity labeling
  • Recombinant baculovirus protein
  • Sf21 cells
  • Tn5 cells

ASJC Scopus subject areas

  • Insect Science
  • Biochemistry, Genetics and Molecular Biology(all)
  • Physiology
  • Physiology (medical)

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    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., & Riddiford, L. M. (1996). Purification and Reassessment of Ligand Binding by the Recombinant, Putative Juvenile Hormone Receptor of the Tobacco Hornworm, Manduca sexta. Archives of Insect Biochemistry and Physiology, 31(4), 371-393.