Characterization of the adipokinetic hormone receptor from the fat body of Manduca sexta

Abstract

A tritium labeled Manduca sexta adipokinetic hormone (M-AKH) was synthesized (pE-L-T-[p 3H]F-T-S-S-W-G-NH 2) (specific activity 27 Ci/mmol) which was fully active in a bioassay. It was used in a filtration based binding assay to characterize the M-AKH receptor from the fat body of M. sexta. Membrane fractions were prepared from fat body and optimal binding conditions were determined. A K d of 7·10 −10 M was determined and the receptor concentration estimated to be 0.5 pmol/mg membrane protein. No receptor binding was found when membranes were prepared from brain, heart or flight muscle of M. sexta or from fat body of the cockroach Blaberus discoidalis. However, specific binding was found with membrane preparations from the pterothoracic ganglion of M. sexta. The membranes from the ganglion had a much smaller number of binding sites than the fat body membranes, however, the binding was specific and observed in each experiment.