Ligand blot analysis of juvenile hormone esterase binding proteins in Manduca sexta L.

Biotinylated recombinant juvenile hormone esterase (JHE) was used for ligan d blotting of proteins from fat body tissue and pericardial athrocytes of M anduca sexta. Proteins were separated by SDS-polyacrylamide gel electrophor esis or by two-dimensional electrophoresis. Eight putative JHE binding prot eins were detected in fat body tissue and in pericardial athrocytes of both M. sexta and Heliothis virescens. The predominant bands were 29, 72, 75, 1 25 and 240 kDa, with minor bands at 50, 80 and 205 kDa. All putative JHE bi nding proteins were present from the second through to the fifth instar lar vae of M. sexta. On wide-range isoelectric focusing, the 29 kDa JHE binding protein separated into three species with isoelectric points of 6.5, 6.6 a nd 6.8. Biotinylated-JHE did not bind recombinant M, sexta-derived juvenile hormone binding protein. The mutant JHE with mutations K29R and K524R bind s weakly to the JHE binding protein P29, relative to binding of wild-type J HE [Shanmugavelu et al., J. Biol. Chem., 275 (2000) 1802-1806]. A similar r eduction in binding was not seen for the 29 kDa binding protein identified here in pericardial athrocytes by ligand blot. This result is discussed. (C ) 2001 Elsevier Science Ltd. All rights reserved.