IDENTIFICATION AND CHARACTERIZATION OF METHYL FARNESOATE BINDING-PROTEINS FROM THE CRAB, CANCER-MAGISTER

Methyl farnesoate (MF) binding proteins were identified in the hemolym ph of male crabs, Cancer magister, using a tritium-labeled photoaffini ty analog of MF, farnesyl diazomethyl ketone (FDK). Crab hemolymph was incubated with [H-3]FDK in the presence of increasing amounts of unla beled MF and the proteins were separated using SDS-PAGE. The associate d 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, hepato pancreas, heart, muscle, epidermis, and Y-organs. Unlabeled MF and FDK were capable of displacing [H-3]FDK from hemolymph MF binding protein s in a dose-dependent way. The apparent dissociation constant (K-d) of each binding protein for MF and FDK was approximately 65 and 100 nM, respectively, as determined by saturation binding studies. A ligand bi nding assay followed by Scatchard analysis was used to determine a mor e accurate apparent K-d value of 145 +/- 10 nM. A single MF binding pe ak was demonstrated when hemolymph samples incubated with [H-3]FDK wer e electrophoresed under nondenaturing conditions. (C) 1997 Academic Pr ess.