Purification of A1 adenosine receptor from rat brain membranes.
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.
Nakata
H
H
Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20892.
D000818:Animals; D001667:Binding, Competitive; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D008970:Molecular Weight; D051381:Rats; D011983:Receptors, Purinergic / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D006882:Hydroxyapatites; D011983:Receptors, Purinergic; D017886:Durapatite