High yield photoaffinity labeling of angiotensin II receptors

The angiotensin II analogues, [Sar1, (4'-N3)Phe8]All and [Sar1, (4'-N3)D-Phe8]All were synthetized and prepared in their iodinated and radioiodinated forms. On rat liver membranes 125I-SarN3PheAll and 125I-SarN3DPheAll labeled a single population of sites (maximal binding capacity, 1.13 +/- 0.16 pmol/mg of protein) with high affinity (dissociation constants of 0.18 +/- 0.05 and 0.37 +/- 0.16 nM, respectively) and high specificity (nonspecific binding less than 10% of total binding for an 0.8 fractional receptor occupancy). 125I-SarN3PheAll and 125I-SarN3DPheAll allowed photoaffinity labeling of liver angiotensin receptors with high efficiency. The yield of photoaffinity labeling was independent of fractional receptor occupancy. 125I-SarN3PheAll and 125I-SarN3DPHeAll allow recovery in a soluble and covalently labeled form of about 40% of the total number of angiotensin receptors from rat liver membranes. For this reason they can be considered as potentially very useful tools for the purification of angiotensin receptors. The material covalently labeled with the antagonist 125I-SarN3DPHeAll was eluted from an Ultrogel ACA34 column as a homogeneous peak (Stoke's radius: 5.5 +/- 0.2 nm). The material covalently labeled with the agonist 125I-SarN3PHeAll was more heterogeneous. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of 125I-SarN3PheAll- and 125I-SarN3DPHeAll-labeled material revealed a single component of 63,000 molecular weight.