Glutathione-binding proteins identified by monoclonal antibodies which depress the behavioral response evoked by glutathione in Hydra

Hydra shows at least 5 components of the behavioral response (R1-R5) which are evoked at different concentrations of S-methylglutathione (GSM). We have prepared several monoclonal antibodies (mAbs), each of which depressed specific responses and visualized specific structures. In this paper, we analyzed molecules participating in the signaling pathway of R5 response with use of three mAbs (J245, J5 and J5/1), all of which depressed the response. A behavioral analysis of the response in the presence of mixtures of these mAbs suggested that J245 and J5 both acted on a component of the receptor-effector system which was not affected by J5/1. Correspondingly, an immunoblotting analysis showed two 220 kDa proteins which reacted with both J245 and J5 but not with J5/1. ELISA analyses also showed that the J245 antigens formed only a portion of the J5 antigens. A labeled major peak was found in the 220 kDa protein fraction by gel permeation HPLC after immunoprecipitation of the J245 antigen photolabeled with [³⁵S]-S-(p-azidophenacyl)-glutathione. Competition of the photolabeling by the ligands GSM and l-glutamate (a competitive inhibitor of the R5 responce) indicated dissociation constants for their binding to the protein of 55 and 90 μM, respectively. These values were consistent with those expected from behavioral experiments. The 220 kDa proteins therefore appear to be candidates for the receptor molecules mediating R5.