The aim of the proposed research is to gain an understanding, on the molecular level, of opiate receptor function in the mammalian nervous system. Methodology currently exists for the solubilization and isolation of active opiate binding polypeptides from both nerve and cell membranes. However, the total specific opiate binding determined for these preparations is a fraction of that originally present in membranes. Moreover, opiate-mediated biological responsiveness, such as the inhibition of adenylate cyclase activity in membranes of the NG108-15 hybrid neurotumor cell line, has not yet been reconstituted from detergent extracts.
The specific aim of this proposal is to develop conditions by which both specific opiate binding and the dose dependent opiate inhibition of adenylate cyclase activity are reconstituted quantitatively from solubilized preparations of NG108-15 cell membranes. Losses of intrinsic opiate binding which occur during solubilization and fractionation will be discriminated from the physical loss of receptor polypeptide by covalently crosslinking specifically bound, radiolabeled opiate to cell membranes and then quantitating the radiolabel following the resolution of polypeptide on SDS-polyacrylamide gels. Solubilization strategies will focus on the use of non-ionic detergents to isolate Delta-opiate receptor from differentiated cells, where it is tightly coupled to adenylate cyclase, and from rapidly growing cells, where it is not. Solubilized receptor will be reconstituted into unilammelar lipid vesicles or into mixed(detergent-lipid-protein)micelles. In the latter case, individual lipid species (or proteins such as Ni) will be added to detergent-receptor mixed micelles so as to regain binding activity. The resulting lipid-protein stoichiometry will be evaluated by chromatographically sizing the mixed micelles. Establishing the unambiguous stoichiometry needed for specific opiate binding will require homogeneous receptor polypeptide. Therefore, the functional receptor, in mixed micellar form, will be fractionated by various chromatographic techniques, including affinity binding to columns of immobilized endorphin and anti-receptor antibody. Differences in specific opiate binding resulting from changes in lipid-protein stoichiometry will provide information about the specificity of opiate drug action. Isolation of the functional opiate binding polypeptide(s) will allow us to obtain detailed chemical information for the actual binding site domain. The development of new drugs that results from an understanding of these requirements is expected to have a tremondous impact in medicine.
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