Purification of a kappa-opioid receptor subtype from frog brain.
A kappa-opioid receptor subtype was purified from a digitonin solubilized preparation of frog brain membranes using affinity chromatography. The affinity resin was prepared by coupling D-Ala2-Leu5-enkephalin to Sepharose-6B matrix. After elution of the receptor by 50 mumol naloxone, the kappa-subtype was separated from the mu- and delta-subtypes by gel permeation chromatography on Sepharose-6B. The purified receptor binds 3,900 pmol [3H]-ethylketocyclazocine per mg protein (a 4,300-fold purification over the membrane-bound receptor) with a KD of 8.3 nM. The purified receptor protein exhibits high affinity for kappa-selective ligands. The purified fraction shows two bands (Mr 65,000 and 58,000) in sodium dodecyl sulfate gel electrophoresis.
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D000818:Animals; D001923:Brain Chemistry; D002850:Chromatography, Gel; D003496:Cyclazocine / Q000031:analogs & derivatives / Q000378:metabolism; D004399:Dynorphins / Q000031:analogs & derivatives / Q000378:metabolism; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine; D004745:Enkephalins / Q000378:metabolism; D016314:Ethylketocyclazocine; D008566:Membranes / Q000032:analysis; D008970:Molecular Weight; D009270:Naloxone / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011759:Pyrrolidines / Q000378:metabolism; D011893:Rana esculenta; D011957:Receptors, Opioid / Q000302:isolation & purification*; D017473:Receptors, Opioid, kappa; D012995:Solubility
D004745:Enkephalins; D010446:Peptide Fragments; D011759:Pyrrolidines; D011957:Receptors, Opioid; D017473:Receptors, Opioid, kappa; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D009270:Naloxone; D016314:Ethylketocyclazocine; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine; D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D004399:Dynorphins; C039666:dynorphin amide (1-10); D003496:Cyclazocine