Identification and characterization of methyl farnesoate binding proteins from the crab, Cancer magister.
Methyl farnesoate (MF) binding proteins were identified in the hemolymph of male crabs, Cancer magister, using a tritium-labeled photoaffinity analog of MF, farnesyl diazomethyl ketone (FDK). Crab hemolymph was incubated with [3H]FDK in the presence of increasing amounts of unlabeled MF and the proteins were separated using SDS-PAGE. The associated fluorogram revealed the presence of two specific MF binding proteins with apparent molecular masses of 34 and 44 kDa. MF binding proteins were not detected in other tissues including testes, eyestalks, hepatopancreas, heart, muscle, epidermis, and Y-organs. Unlabeled MF and FDK were capable of displacing [3H]FDK from hemolymph MF binding proteins in a dose-dependent way. The apparent dissociation constant (Kd) of each binding protein for MF and FDK was approximately 65 and 100 nM, respectively, as determined by saturation binding studies. A ligand binding assay followed by Scatchard analysis was used to determine a more accurate apparent Kd value of 145 +/- 10 nM. A single MF binding peak was demonstrated when hemolymph samples incubated with [3H]FDK were electrophoresed under nondenaturing conditions.
Tamone
S L
SL
Bodega Marine Laboratory, University of California, Bodega Bay, California, 94923, USA.
Prestwich
G D
GD
Chang
E S
ES
D000345:Affinity Labels / Q000032:analysis / Q000378:metabolism; D000818:Animals; D003386:Brachyura / Q000378:metabolism*; D002352:Carrier Proteins / Q000097:blood / Q000378:metabolism*; D003978:Diazomethane / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000662:veterinary; D005204:Farnesol / Q000031:analogs & derivatives / Q000032:analysis / Q000378:metabolism; D005231:Fatty Acids, Unsaturated / Q000737:chemistry / Q000378:metabolism*; D006458:Hemolymph / Q000737:chemistry / Q000378:metabolism*; D008297:Male; D011869:Radioligand Assay / Q000662:veterinary; D014316:Tritium
D000345:Affinity Labels; D002352:Carrier Proteins; D005231:Fatty Acids, Unsaturated; D014316:Tritium; C032429:methyl farnesoate; D005204:Farnesol; D003978:Diazomethane; C067747:farnesyl diazomethyl ketone