Cell-free synthesis of opiate binding sites

A procedure is described for the detection of opiate binding sites synthesized during in vitro translation of various mRNA preparations. RNA were isolated from membrane bound polysomes which were prepared from NG 108-15 hybridoma, C6BU1 glioma cells, as well as from N18TG2, NB2aAg and NB41A3 neuroblastoma cells. Polyadenylated [poly(A)(+)] RNA were purified, translated in vitro in a rabbit reticulocyte lysate and the translation products assayed for their ability to bind [(3)H] bremazocine. Bound and free ligands were separated by column chromatography. After translation of poly(A)(+) RNA obtained from NG 108-15 cells we demonstrated a stereospecific, saturable binding of [(3)H]bremazocine (displaced by levorphanol and not by dextrorphan) with a K(d) of 2.4 +/- 1.0 nM. The total amount of opiate binding sites synthesized was 6.2 +/- 0.5 fmol per ?g of poly(A)(+) RNA. Opiate binding sites were undetectable at zero time and a plateau was reached after translation had proceeded for 20 min. Five time less opiate binding sites were synthesized when the poly(A)(+) RNA purified from N18TG2 neuroblastoma cells were used under the same experimental conditions. There was no detectable binding of opiate ligands with poly(A)(+) RNA obtained from C6BU1 glioma cells, NB2aAg or NB41A3 neuroblastoma cells.