Abstract
The A1 adenosine receptor from rat brain membranes has been purified about 50,000-fold to apparent homogeneity by sequential use of affinity chromatography on immobilized xanthine amine congener-agarose, hydroxylapatite chromatography, and reaffinity chromatography. The overall yield starting from the membranes was approximately 4%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified preparation gave a broad single band of an apparent molecular weight of 34,000 either by silver staining or autoradiogram after radioiodination. The purified receptor bound approximately 24 nmol of 8-cyclopentyl-1,3-[3H]dipropylxanthine/mg of protein with a dissociation constant of 1.4 nM. This maximum specific binding value is consistent with the expected theoretical specific activity (29.4 nmol/mg) for a protein with a molecular mass of 34,000 daltons if it is assumed that there is one ligand-binding site/receptor molecule. Affinity-labeling experiments using [3H]p-phenylenediisothiocyanate-xanthine amine congener showed that the Mr = 34,000 protein band contained the ligand-binding sites. The purified receptor gave a typical A1 adenosine receptor pharmacological specificity similar to that of unpurified receptor preparations.
Highlights
Molecular weight of 34,000 either by silver staining or autoradiogram after radioiodination
The affinity chromatography using XAC as animmobilizedligand has been describedand characterized recently (13).In this study, the procedure was modified for large scale purification
It should be noted that the adenosine antagonist, CPT, present in the eluates of the XAC-agarose chromatography was removed fromthe receptor preparation during the hydroxylapatite chromatography
Summary
The AI adenosine receptor from rat brain membraanneesfficient methodof purification. Has been purified about 50,000-fold to apparent ho- reported partial purification of the rat A1 adenosine receptor mogeneity by sequential useof affinity chromatogra- by affinity chromatography using agarose immobilized with phy on immobilized xanthine amine congener-agarosex,anthine amine congener (XAC),' a potent A1antagonist (14). Hydroxylapatite Chromatography, andreaffinity chro- In thispaper, an improvement of the affinity chromatography matography. Theoverall yield starting from the mem- and the purification of Al adenosine receptor from rat brain braneswas approxim.ately4%.Sodium dodecyslulfate- membranes to apparenthomogeneity are reported. Polyacrylamide gel electrophoresis of thepurified preparation gave a broad single band of an apparent Theoverall yield starting from the mem- and the purification of Al adenosine receptor from rat brain braneswas approxim.ately4%.Sodium dodecyslulfate- membranes to apparenthomogeneity are reported. polyacrylamide gel electrophoresis of thepurified preparation gave a broad single band of an apparent
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