Purification and characterization of the mu opiate receptor from rat brain using affinity chromatography.

Publisher Website
Google Scholar
Abstract
Opiate receptors were solubilized from rat neural membranes and purified 500-fold (relative to the crude solubilized extract) by affinity chromatography. Active receptors were solubilized by using 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), a zwitterionic derivative of cholic acid. Affinity chromatography was carried out using Affi-Gel 401, a sulfhydryl derivative of agarose to which hybromet, a newly synthesized opioid ligand with high affinity for the .mu. receptor, had been attached. Scatachard analysis of [3H]etorphine binding to the purified receptor revealed a single class of high-affinity sites (Kd = 1.4 nM; Bmax = 2800 fmol/mg of protein). Half-maximal binding was achieved at .apprxeq. 1 nM. Activity was markedly inhibited by protein modifying reagents, findings which suggest that the sites are proteinaceous. Opiate binding activity was also inhibited by GTP. Electrophoresis of the purified material under denaturing conditions revealed 3 subunits of MW 94,000, 44,000 and 35,000. The inhibitory guanyl nucleotide binding protein (Ni) implicated in opiate action was comprised of 2 subunits of MW 42,000 and 35,000. The opiate receptor may be an aggregate of multiple protein components that may include a guanyl nucleotide binding protein.