Purification and characterization of the mu opiate receptor from rat brain using affinity chromatography.
Opiate receptors have been 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. Scatchard 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 approximately equal to 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 the guanyl nucleotide GTP. Electrophoresis of the purified material under denaturing conditions revealed three subunits of molecular weights 94,000, 44,000, and 35,000. The inhibitory guanyl nucleotide binding protein (Ni) implicated in opiate action has been shown to be comprised of two subunits of molecular weights 42,000 and 35,000. Thus, the opiate receptor may be an aggregate of multiple protein components that may include a guanyl nucleotide binding protein.
Maneckjee
R
R
Zukin
R S
RS
Archer
S
S
Michael
J
J
Osei-Gyimah
P
P
D000818:Animals; D001923:Brain Chemistry; D002793:Cholic Acids; D002846:Chromatography, Affinity / Q000379:methods; D004090:Dihydromorphine / Q000378:metabolism; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine; D005048:Etorphine / Q000378:metabolism; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011957:Receptors, Opioid / Q000302:isolation & purification* / Q000378:metabolism; D017450:Receptors, Opioid, mu
D002793:Cholic Acids; D011480:Protease Inhibitors; D011957:Receptors, Opioid; D017450:Receptors, Opioid, mu; D005048:Etorphine; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; D004090:Dihydromorphine; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate