IDENTIFICATION AND PARTIAL CHARACTERIZATION OF RECEPTORS FOR ALLATOSTATINS IN BRAIN AND CORPORA ALLATA OF THE COCKROACH DIPLOPTERA-PUNCTATA USING A BINDING ASSAY AND PHOTOAFFINITY-LABELING

We have developed both an in vitro binding assay and a photoaffinity labeling assay to demonstrate and partially characterize putative receptors for allatostatins in brain and in corpora allata of Diploptera punctata. Isolated brain membranes were photoaffinity labeled with I-125-RYBPA (photoaffinity analogue of dip-allatostatin 5). Following labeling with I-125-RYBPA, SDS-PAGE and autoradiography revealed the presence of a putative receptor (37 kDa) for dip-allatostatin 5 and dip-allatostatin 7. Specific labeling was demonstrated by dose-dependent competition with either dip-allatostatin 5 or dip-allatostatin 7. The in vitro binding assay indicated that the receptor for dip-allatostatin 5 had a K-d of (9.0 +/- 0.9). 10(-10) M and B-max of 2.2 +/- 0.3 pmol/mg membrane protein. For dip-allatostatin 7, two K-d values of (1.5 +/- 0.1). 10(-9) M and (3.8 +/- 0.3). 10(-9) M were obtained, with B-max values of 7.2 +/- 0.7 pmol/mg membrane protein and 11.4 +/- 1.0 pmol/mg membrane protein respectively. This indicates that there were probably two putative receptor sites for dip-allatostatin 7 although only one band was observable following photoaffinity labeling. Binding was saturable, specific and reversible. Using the in vitro binding assay, the K-d of the putative receptor in CA for dip-allatostatin 7 was shown to be (7.2 +/- 0.9). 10(-10) M.