Conformational change of the haemolymph juvenile-hormone-binding protein from Galleria mellonella (L).
A juvenile-hormone-binding protein (juvenile-hormone carrier), isolated from Galleria mellonella haemolymph, was treated with trypsin, chymotrypsin, carboxypeptidase A and subtilisin. Among these enzymes, only subtilisin was able to affect juvenile-hormone-binding activity of this protein. With SDS/PAGE it was shown that juvenile-hormone-binding protein, a 32-kDa peptide, is first slowly converted into a 30-kDa molecule, then into two or three smaller-molecular-mass species (20-25 kDa), which in turn were further digested to small peptides undetectable in PAGE. The 30-kDa peptide has a 2.4-times-higher dissociation constant for juvenile hormone than the native protein. No binding activity was detected for 20-25-kDa peptides. The rate of proteolysis of juvenile-hormone-binding protein was decreased by more than twofold in the presence of hormone, however, the overall cleavage pattern was unchanged. Under non-denaturing conditions, free binding-protein molecules could be separated from juvenile-hormone-binding-protein complex due to a slower electrophoretic mobility of the complex. As judged from ultracentrifugation and cross-linking experiments, binding of the hormone to its haemolymph carrier does not induce formation of oligomers, but shifts the sedimentation coefficient from 2.30S to 2.71S. It is concluded that juvenile-hormone binding induces a conformational transition of its carrier protein. This hormone-induced change might have a physiological significance for signal transmission.
Wieczorek
E
E
Division of Biochemistry, Technical University of Wrocław, Poland.
Kochman
M
M
D000818:Animals; D002268:Carboxypeptidases / Q000737:chemistry; D043422:Carboxypeptidases A; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002918:Chymotrypsin / Q000737:chemistry; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006458:Hemolymph / Q000737:chemistry*; D006868:Hydrolysis; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000378:metabolism*; D007915:Lepidoptera; D011487:Protein Conformation; D013381:Subtilisins / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry; D014461:Ultracentrifugation
D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D019476:Insect Proteins; D007605:Juvenile Hormones; C035606:juvenile hormone-binding protein, insect; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D013381:Subtilisins; D002918:Chymotrypsin; D014357:Trypsin