title: Photoaffinity labeling adenosine A1 receptors with an antagonist 125I-labeled aryl azide derivative of 8-phenylxanthine.
abstract: We have derivatized a series of 125I-labeled 8-phenylxanthines with photoactive aryl azide groups on the 1- or 3-position of the xanthine ring. A 3-azidophenethyl derivative was found to be optimal for use as an antagonist photoaffinity label for adenosine A1 receptors. Following photoactivation, radioactivity was covalently and specifically incorporated into a 34,000-dalton and, to a lesser extent, into a 24,000-dalton polypeptide of rat brain membranes. Photoincorporation into both polypeptides was competitively inhibited by adenosine analogues with a potency order typical of adenosine A1 receptors, but the 24,000-dalton polypeptide bound both agonists and antagonists with lower affinity than the 34,000-dalton polypeptide. Specific photolabeling of receptors in brain membranes of rat, guinea pig, dog, and cow did not show any variation in the 34,000-dalton adenosine receptor binding subunit. The adenosine agonist photoaffinity label [125I]N6-(4-azido-3-iodobenzyl)adenosine also specifically photolabeled the 34,000-dalton polypeptide, but photoincorporation of the agonist was less efficient than the antagonist and, unlike the antagonist, was greatly reduced by guanosine 5'-(beta,gamma-imidotriphosphate). The results indicate that the antagonist photoaffinity label may be more useful than agonists particularly for labeling uncoupled receptors.
authors.affiliation: Department of Physiology, University of Virginia School of Medicine, Charlottesville 22908.
authors.forename: C Q
authors.initials: CQ
authors.lastname: Earl
authors.forename: A
authors.initials: A
authors.lastname: Patel
authors.forename: R H
authors.initials: RH
authors.lastname: Craig
authors.forename: S M
authors.initials: SM
authors.lastname: Daluge
authors.forename: J
authors.initials: J
authors.lastname: Linden
mesh_terms: D000345:Affinity Labels / Q000138:chemical synthesis / Q000378:metabolism*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis / Q000378:metabolism*; D001921:Brain / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D006168:Guinea Pigs; D007457:Iodine Radioisotopes; D007553:Isotope Labeling; D008566:Membranes / Q000378:metabolism; D008970:Molecular Weight; D010777:Photochemistry; D011687:Purines / Q000138:chemical synthesis / Q000378:metabolism*; D051381:Rats; D011983:Receptors, Purinergic / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D000345:Affinity Labels; D001386:Azides; D007457:Iodine Radioisotopes; D011687:Purines; D011983:Receptors, Purinergic; C055430:BWA 947U