COVALENT MODIFICATION OF JUVENILE-HORMONE BINDING-PROTEINS BY PHOTOAFFINITY-LABELING - AN UNEXPECTED GEL SHIFT EFFECT

The 32 kD juvenile hormone binding protein (JHBP) and two 80 kD protei ns in larval Manduca sexta hemolymph were labeled with [H-3]FDK, a pho toaffinity analog of methyl farnesoate (MF). The labeling could be com pletely displaced by a 30-fold excess of either MF or JH II, demonstra ting that [H-3] FDK binds specifically to the JH binding sites of the 32 kD JHBP and the 80 kD proteins. In addition, a high molecular-mass protein was labeled with [H-3]FDK; labeling could be displaced by exce ss MF but not by JH II, demonstrating the selectivity in binding MF. T he 32 kD JHBP also appeared to weakly bind the potent juvenoid, methop rene, at the JH binding site. Covalent modification by [H-3]FDK induce d a change in the apparent size and the isoelectric point of the JHBP. These changes were not induced by substrate alone, nor by UV irradiat ion alone. The same effect was also observed during labeling with [H-3 ]MDK, an analog of methoprene. These data provide an important caveat for anticipating artifactual changes of protein properties during chem ical or photochemical affinity labeling experiments. The molecular dim ensions of [H-3]FDK more closely resemble those JH II than those of [H -3] EHDA, a photoactivatable analog of JH Il. We suggest that covalent modification by a diazoketone photolabel involves a hydrophilic amino acid important in the recognition of the ester group of JH. (C) 1994 Wiley-Liss, inc.