Affinity chromatography in purification of A 1 adenosine receptors

Abstract

Purification of A 1 adenosine receptor of rat brain membranes was performed using a newly developed affinity gel employing xanthine amine congener (XAC) as an immobilized ligand. The A 1 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 A 1 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 M r = 34 000. This band was also shown to be specifically labelled with an affinity labelling reagent for A 1 adenosine receptors. This purification method was also applicable for the complete purification of A 1 adenosine receptors from rat testis and human brain membranes.