Identification of opioid-binding materials of rat brain.
Digitonin-solubilized opioid receptors from rat brain were purified with an affinity resin, AH-Sepharose coupled with [D-Ala2, D-Leu5]enkephalin (DADLE). Radioreceptor binding assay showed that the purified materials had specific opioid-binding activity of 310 pmol/mg protein on DADLE binding. Analyses by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) revealed that the materials were rich in two polypeptides; the major component had a molecular weight of 62000-64000. To establish the materials responsible for binding opiates, the purified materials were cross-linked with 125I-labeled beta-endorphin using bis[2-(succinimidooxycarbonyloxy)-ethyl]sulfone as a cross-linker. The molecular weight of 62000-64000, the major band of the purified materials on SDS-PAGE, agreed closely with that determined by the cross-linking experiment. The results suggest that the purified materials contained opioid-binding materials (opioid receptors).
Fujioka
T
T
Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Hyogo, Japan.
Inoue
F
F
Kuriyama
M
M
Miura
Y
Y
Higashijima
T
T
D000818:Animals; D001923:Brain Chemistry; D002417:Cattle; D004743:Enkephalin, Leucine / Q000031:analogs & derivatives / Q000378:metabolism; D016308:Enkephalin, Leucine-2-Alanine; D051381:Rats; D011957:Receptors, Opioid / Q000032:analysis / Q000302:isolation & purification*
D011957:Receptors, Opioid; D004743:Enkephalin, Leucine; D016308:Enkephalin, Leucine-2-Alanine