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Affinity chromatography of the bovine cerebral cortex A 1 adenosine receptor
Author(s) -
Olah Mark E.,
Jacobson Kenneth A.,
Stiles Gary L.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81555-8
Subject(s) - affinity chromatography , chemistry , receptor , adenosine receptor , affinity label , adenosine , affinity labeling , size exclusion chromatography , biochemistry , photoaffinity labeling , chromatography , agonist , enzyme
An approximate 140‐fold purification of the A 1 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 A 1 adenosine receptor antagonist. As assessed by [ 3 h]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1protein. Interaction of solubilized A 1 adenosine receptors with the XAC‐Affi‐Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [ 3 h]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 ± 22protein 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 A 1 adenosine receptor: R ‐PIA > S ‐PIA >62; NECA. In purified preparations, the photoaffinity probe [ 125 1]PAPAXAC‐SANPAH specifically labelled a protein of molecular mass 38000 which has previously been shown to be the A 1 adenosine receptor binding subunit.