Purification, characterisation and titre of the haemolymph juvenile hormone binding proteins from Schistocerca gregaria and Gryllus bimaculatus.
Juvenile hormone binding proteins (JHBPs) were extracted from the haemolymph of adult desert locusts, Schistocerca gregaria, and Mediterranean field crickets, Gryllus bimaculatus. The JHBPs were purified by polyethyleneglycol precipitation, filtration through molecular weight cut off filters and chromatography on a HiTrap heparin column. The juvenile hormone (JH) binding activity of the extracts was measured using a hydroxyapatite assay and the purification progress was monitored by native gel chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The haemolymph JHBPs of both insects are hexamers composed of seemingly identical subunits. The JHBP of the locust has a native Mr of 480 kDa with subunits of 77 kDa, whereas the JHBP of the cricket has a Mr of 510 kDa with subunits of 81 kDa. The locust JHBP binds JH III with moderate affinity (KD = 19 nM). Competition for binding of JH II and JH I was about 2 and 5 times less, respectively. The cricket JHBP also has a moderate affinity for JH III (KD = 28 nM), but surprisingly, competition for binding of JH II was equal to that of JH III and JH I competed about 3 times higher. No sequence information was obtained for the locust JHBP, but the N-terminal sequence of the cricket JHBP shows ca. 56% sequence homology with a hexamerin from Calliphora vicina. Antisera raised against the purified JHBPs were used to measure age- and sex-dependent changes in haemolymph JHBP titres and to confirm that the JHBPs of both species are immunologically different.
Tawfik
Amer I
AI
Department of Zoology/Entomology, Faculty of Science, Assiut University, Assiut 71516, Egypt.
Kellner
Roland
R
Hoffmann
Klaus H
KH
Lorenz
Matthias W
MW
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D006110:Grasshoppers / Q000737:chemistry* / Q000378:metabolism; D006135:Gryllidae / Q000737:chemistry* / Q000378:metabolism; D006458:Hemolymph / Q000737:chemistry; D019476:Insect Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017386:Sequence Homology, Amino Acid; D012717:Sesquiterpenes / Q000378:metabolism
D002352:Carrier Proteins; D019476:Insect Proteins; D012717:Sesquiterpenes; C035606:juvenile hormone-binding protein, insect; C036585:juvenile hormone III