Identification of opioid-binding materials of rat brain |
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Authors: | T Fujioka F Inoue M Kuriyama Y Miura T Higashijima |
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Institution: | Faculty of Pharmaceutical Sciences, Mukogawa Women's University, Hyogo, Japan. |
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Abstract: | 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 bis2-(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). |
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