Juvenile hormone binding proteins from the epidermis of Galleria mellonella

Abstract

Juvenile hormone binding proteins (JHBP) have been detected in cytosol of larval and pupal epidermis of Galleria mellonella. Juvenile hormone (JH) binding activity changes during insect development, reaching a maximum after each ecdysis. Using density gradient centrifugation, three distinct peaks of JHBP have been detected: 8.8–9.2 S, 4.2–4.6 S, and 3 S. Despite molecular mass heterogeneity of the JHBPs, only one class of JH binding sites has been detected. The equilibrium dissociation constants ( K d) of the larval JHBP for the natural (10 R, 11 S) JHs and their enantiomers are: K d, (10 R, 11 S)-JH I = 24 ± 9 nM; K d, (10 S, 11 R)-JH I = 51 ± 15 nM; K d, (10 R, 11 S)-JH II = 9.7 ± 2.0 nM, K d, (10 S, 11 R)-JH II = 18 ± 3 nM. From competitive binding assays using 3H-labeled (10 R, 11 S)-JH II, it was found that racemic JH III and the JH analog (10 R, 11 S)-12-iodo-JH I are bound with affinity close to the unnatural (10 S, 11 R) enantiomer of JH I. Juvenoid R 394 (a methoprene analog) is bound to JHBP with an affinity 100-fold lower than (10 R, 11 S)-JH II, whereas binding of the epoxygeranylphenyl ether juvenoid R 20458 is not detectable. Addition of 0.1% (w/v) Zwittergent 3–10 to the epidermal cytosol causes an increase of JH binding activity by 2–10-fold in penultimate and ultimate instars, respectively. This zwitterionic N-alkyl sulfobetaine detergent apparently converts a high molecular mass JHBP species into a 65-kDa component, but does not change the stereospecificity or the affinity of JHBP towards the JH homologs or analogs.