Affinity purification and binding analysis of the hemolymph juvenile hormone binding protein from Manduca sexta.
A high-affinity juvenile hormone binding protein was purified from the hemolymph of the tobacco hornworm, Manduca sexta, employing ammonium sulfate precipitation and affinity and size-separation chromatography. The naturally occurring enantiomer of juvenile hormone III (10R) was converted to juvenile hormone III acid and then covalently attached to aminohexyl-Sepharose 4B. Hemolymph from early fifth stadium (60 h postecdysis) larvae was used as the source of hJHBP. The yield of hJHBP was approximately 25% of the starting material, with 3.5 mg of highly purified, biologically active hJHBP recovered from 100 mL of hemolymph. Binding parameters were examined using equilibrium dialysis and highly purified, enantiomerically correct juvenile hormone I and II and racemic JH III. The equilibrium dissociation constants for juvenile hormone I and II were approximately 6 x 10(-10) M at 4 degrees C, while racemic juvenile hormone III displayed an equilibrium dissociation constant of 1.9 x 10(-9) M. At 25 degrees C the equilibrium dissociation constant for juvenile hormone I was 1.6 x 10(-9) M. Half-times of dissociation were also determined for the three homologs. The half-time of dissociation was 30 s for juvenile hormone I, 20 s for juvenile hormone II, and 13 s for juvenile hormone III at either 4 or 25 degrees C. Using the new equilibrium dissociation constants, we calculate that better than 99% of the circulating juvenile hormone titer may be bound to this hemolymph protein.
Park
Y C
YC
Department of Entomology, University of Wisconsin-Madison 53706.
Tesch
M J
MJ
Toong
Y C
YC
Goodman
W G
WG
D000818:Animals; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity; D006458:Hemolymph / Q000737:chemistry*; D019476:Insect Proteins; D007605:Juvenile Hormones / Q000378:metabolism*; D007700:Kinetics; D009036:Moths / Q000737:chemistry*; D011485:Protein Binding; D013237:Stereoisomerism
D002352:Carrier Proteins; D019476:Insect Proteins; D007605:Juvenile Hormones; C035606:juvenile hormone-binding protein, insect