Cross-linking is a relatively recent biochemical strategy for covalently affixing reversible ligands to their recognition molecules for subsequent electrophoretic analysis. (125I)-Tyr27- beta-endorphin (prepared as originally described by Smythe and co-workers) binds stereospecifically to rat brain membranes. While some studies have suggested that the beta-endorphin receptor is a unique """"""""epsilon"""""""" opiate receptor, a larger body of evidence suggest that beta-endorphin has high affinity for most if not all of the opiate receptors types and subtypes. Cross-linking opiate receptors from different tissue sources can potentially reveal much information about the molecular basis of apparent opiate receptor heterogeneity. Cross-linking, however, only fixes 1% of the bound trace and SDS-PAGE while exquisitely sensitive, can fail to reveal substantial inter-molecular differences. Cross- linking was performed with the homo bi-functional reagent Disuccinimidyl Suberate (DSS). The iodinated cross-linking products of Tetrahymena, leech CNS, and rat brain membranes (both type 1 and type 2 conditions) appeared indistinguishable on SDS-PAGE gel with major cross-linking products at 58K and 100- 110K. The strong cross-linking bands produced by incubation in the presence of the inactive opiate ((+)-naloxone) was completely abolished by the same (10-6M) concentration of its active isomer (-)-naloxone. Although we have thus far failed to distinguish between opiate receptors from a mammal, an invertebrate, and a unicellular organism, we continue to explore various conditions of binding, and electrophoresis, (e.g., reduced and unreduced) to examine possible receptor differences, both intra and inter species. Electrophoresis of proteolytic digests of cross-linked bands will be performed as a particularly sensitive method for distinguishing heterogeneity. Thus far, our cross-linking experiment suggest that the recognition molecule (the opiate receptor) which binds all opiate alkaloids and peptides is stable across evolution. As proposed, apparent physiological receptor heterogeneity is due to coupling to other membrane components.
