5'-N-ethylcarboxamido [3H] adenosine binding sites of mouse P815 mastocytoma cell membranes: solubilization and partial purification by affinity chromatography.
A 5'-N-ethylcarboxamido[3H]adenosine ([3H]NECA) binding site of mouse mastocytoma P815 cell membranes has been purified approximately 100-fold by affinity chromatography. This adenosine binding site, which has a similar specificity to that of the A2 adenosine receptor, was absorbed on NECA-linked Sepharose 6B and eluted with NECA. The adsorption of the [3H]NECA binding site to the affinity matrix was specifically blocked by NECA. The [3H]NECA binding site bound on the affinity matrix was also specifically eluted by NECA. This affinity matrix adsorbed approximately 90% of the digitonin-solubilized [3H]NECA binding activity applied, and after the gel was washed, 30-50% of the adsorbed binding activity could be eluted with 500 microM NECA with specific binding activity of 50-70 pmol/mg of protein. The affinity-purified [3H]NECA binding site retained the same ligand binding specificities as the original membrane preparation. The results indicate that the NECA-Sepharose Sepharose 6B should provide a powerful tool for the eventual purification of [3H]NECA binding sites of P815 cell membranes.
Nakata
H
H
Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, MD 20892.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D012515:Mast-Cell Sarcoma / Q000378:metabolism*; D051379:Mice; D011983:Receptors, Purinergic / Q000302:isolation & purification*; D014407:Tumor Cells, Cultured / Q000378:metabolism
D011983:Receptors, Purinergic; D019830:Adenosine-5'-(N-ethylcarboxamide); D000241:Adenosine