This is a proposal to study the heterogeneity of opiate-receptor interaction, with particular emphasis on identifying and characterizing the distinct sites or classes of analgesic binding sites, believed to exist for alkaloids and peptides. A protein-lipid model of the opiate receptor is proposed which has two binding sites, one for enkephalin on the protein portion and one for alkaloids on the lipid portion, with B-endorphin interacting with both. The following types of experiments are used to test this model: 1) The effect of intracerebroventricularly (icv) injections of various enkephalins and endorphin fragments on morphine analgesia in mice; preliminary experiments have already shown that certain peptides potentiate morphine analgesia at doses at which the peptide alone has no effect, suggesting that they act at a different site from that of the alkaloid; 2) Comparison of the ability of various unlabelled opiates to displace binding of radioactive alkaloid or peptide to brain subcellular fractions, with particular attention to various peptides which have not been studied this way before; 3) Biochemical characterization of the binding sites, both by testing the effect of specific protein- or lipid-modifying reagents on in vitro binding, and directly by solubilizing binding material in Brij 36-T and further analyzing; 4) Circular dichroism measurements of B-endorphin and various endorphin fragments; we have shown in preliminary experiments that B-endorphin, though normally existing as a random chain in solution, forms alpha-helical structure in the presence of lipid, which may be necessary for receptor interaction. These experiments should allow us: 1) to conform the existence of separate binding sites for alkaloids and peptides; 2) to identify distinct sites that may exist for different types of alkaloids or different types of peptides; 3) to gain some insight into the chemical nature of these sites, particularly with respect to whether they are located on lipid or protein, and whether they are on distinct molecules; and 4) to begin purification of some of these receptors.
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