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 125I-RYBPA (photoaffinity analogue of dip-allatostatin 5). Following labeling with 125I-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 Kd of (9.0 +/- 0.9).10(-10) M and Bmax of 2.2 +/- 0.3 pmol/mg membrane protein. For dip-allatostatin 7, two Kd values of (1.5 +/- 0.1).10(-9) M and (3.8 +/- 0.3).10(-9) M were obtained, with Bmax 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 Kd of the putative receptor in CA for dip-allatostatin 7 was shown to be (7.2 +/- 0.9).10(-10) M.