Purification and partial amino acid sequence of a mu opioid receptor from rat brain.
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.
Eppler
C M
CM
Agricultural Research Division, American Cyanamid Co., Princeton, New Jersey 08543.
Hulmes
J D
JD
Wang
J B
JB
Johnson
B
B
Corbett
M
M
Luthin
D R
DR
Uhl
G R
GR
Linden
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001710:Biotin; D001923:Brain Chemistry; D002462:Cell Membrane / Q000737:chemistry; D002846:Chromatography, Affinity; D018076:DNA, Complementary; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D006031:Glycosylation; D007457:Iodine Radioisotopes; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D017450:Receptors, Opioid, mu / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility; D001615:beta-Endorphin
D018076:DNA, Complementary; D007457:Iodine Radioisotopes; D017450:Receptors, Opioid, mu; D001615:beta-Endorphin; D001710:Biotin; D019204:GTP-Binding Proteins