Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor

Abstract

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.

Fig.1

[³H]XAC saturation curves in (A) solubilized bovine brain membranes and (B) XAC-Affi-Gel-purified preparation. Membrane solubilization, affinity chromatography and binding assays were performed as described in section 2. Non-specific binding was defined with 10 µM R-PIA or 5 mM theophylline. Gpp(NH)p (10 µM) was present in all assay tubes. These plots are representative of 5 similar experiments.

Fig.2

Agonist competition for [³H]XAC binding in (A) solubilized bovine brain membranes and (B) XAC-Affi-Gel-purified preparation. Membrane solubilization, affinity chromatography and binding assays were performed as described in section 2. The final concentration of [³H]XAC was ~ 0.5 nM in both assays. These plots are representative of 3 similar experiments.

Fig.3

Photoaffinity labelling of XAC-Affi-Gel-purified A₁ adenosine receptor with [¹²⁵I]PAPAXAC-SANPAH. Chromatography and photoaffinity labelling were performed as described in section 2. Labelling was performed in the absence (control) or presence of 5 mM theophylline. Following labelling, the sample was solubilized in SDS and electrophoresed on a 12% acrylamide gel. The positions of iodinated molecular mass standards are shown to the left.