Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor.
An approximate 140-fold purification of the A1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi-Gel 10 coupled through an amide linkage to XAC, a high-affinity A1 adenosine receptor antagonist. As assessed by [3H]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 pmol/mg protein. Interaction of solubilized A1 adenosine receptors with the XAC-Affi-Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [3H]XAC binding in the material eluted from the column with R-PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 +/- 22 pmol/mg protein with typically 5-15% of the bound receptor recovered. The purified receptor displayed high-affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A1 adenosine receptor: R-PIA greater than S-PIA greater than NECA. In purified preparations, the photoaffinity probe [125I]PAPAXAC-SANPAH specifically labelled a protein of molecular mass 38,000 which has previously been shown to be the A1 adenosine receptor binding subunit.
Olah
M E
ME
Department of Medicine Cardiology, Duke University Medical Center, Durham, NC 27710.
Jacobson
K A
KA
Stiles
G L
GL
D000818:Animals; D002417:Cattle; D002462:Cell Membrane / Q000032:analysis; D002540:Cerebral Cortex / Q000032:analysis*; D002846:Chromatography, Affinity; D008970:Molecular Weight; D011983:Receptors, Purinergic / Q000302:isolation & purification*
D011983:Receptors, Purinergic