Purification and partial amino acid sequence of a mu opioid receptor from rat brain

• Published in: The Journal of biological chemistry Vol. 268; no. 35; pp. 26447 - 26451
• Main Authors: Eppler, C M, Hulmes, J D, Wang, J B, Johnson, B, Corbett, M, Luthin, D R, Uhl, G R, Linden, J
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 15.12.1993; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Base Sequence; beta-Endorphin; Biological and medical sciences; Biotin; Brain Chemistry; Cell Membrane - chemistry; Cell receptors; Cell structures and functions; Chromatography, Affinity; DNA, Complementary; Electrophoresis, Polyacrylamide Gel; Fundamental and applied biological sciences. Psychology; Glycosylation; GTP-Binding Proteins - metabolism; Iodine Radioisotopes; Male; Miscellaneous; Molecular and cellular biology; Molecular Sequence Data; Rats; Receptors, Opioid, mu - chemistry; Receptors, Opioid, mu - isolation & purification; Solubility; Ligand binding; Membrane receptor; Vertebrata; Mammalia; Purification; Rat; Rodentia; Primary structure; Molecular cloning; Opiate receptor μ; Brain (vertebrata)
• DOI: 10.1016/S0021-9258(19)74335-X
• ISSN: 0021-9258

A rat brain opioid receptor protein was isolated by binding [epsilon-biotinyl-Lys32] beta-endorphin to membranes, solubilizing the receptor-ligand (R.L) complex with deoxycholate-lysophosphatidylcholine and purifying on immobilized streptavidin and wheat germ agglutinin. The purified glycoprotein had a molecular mass of 60-70 kDa. Recovery of this protein was blocked by the nonselective opioid antagonist naloxone and the highly mu-selective agonist [D-Ala2,N-methyl-Phe4,Glyol5]-enkephalin but not by the highly delta-selective agonist [D-Pen2,4'-Cl-Phe4,D-Pen5]enkephalin when these compounds were added as competitors at the binding step. The 60-70-kDa receptor protein co-purified through the streptavidin column with 40-kDa protein recognized by anti-Gi alpha antibodies. GTP and Na+ influenced dissociation of the solubilized R.125I-L complex and elution of the receptor and G protein from streptavidin in fashions consistent with the pharmacology of mu-opioid receptors. A 23-amino acid residue sequence from the purified receptor differs at 4 positions from a similar sequence in the murine delta-opioid receptor and is encoded within a novel rat brain cDNA isolated by polymerase chain reaction with oligonucleotide primers related to the murine delta-opioid receptor gene.