Affinity chromatography in purification of A1 adenosine receptors

Purification of A1 adenosine receptor of rat brain membranes was performed using a newly developed affinity gel employing xanthine amine congener (XAC) as an immobilized ligand. The A1 adenosine receptor was solubilized with digitonin-cholate from brain membranes and then purified by a sequential use of affinity chromatography on XAC-agarose, hydroxyapatite chromatography and reaffinity chromatography on XAC-agarose. The A1 adenosine receptor was purified ca. 45,000-fold with a yield of 5%. The final receptor preparation gave a single broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a Mr approximately 34,000. This band was also shown to be specifically labelled with an affinity labelling reagent for A1 adenosine receptors. This purification method was also applicable for the complete purification of A1 adenosine receptors from rat testis and human brain membranes.