Purification and partial characterization of a novel, non‐AT1, non‐AT2 binding site for angiotensins in the rat brain

Abstract

The structure of the novel, non‐AT1, non‐AT2 binding site for angiotensins (Ang) (Brain Res. 2007, 1143: 83) is still unknown. We used Sar1‐p‐benzoyl‐L‐Phe8‐Ang II to specifically photolabel this protein in rat brain membranes. Photolabeled membrane pellet extracts were resolved by 7.5% SDS‐PAGE. The radioactive peak in the "Total" binding samples was 75‐80 kDa with negligible amounts of radioactivity at this point in corresponding "Non‐specific" samples. Gel cuts from the radioactive band from the "Total" samples were combined and radioactivity was extracted. The molecular weight of the semipurified binding site was not altered by glycopeptidase‐F indicating that the protein is not N‐glycosylated. Neither GTPgammaS nor GppNHP (at 50 µM) alter Ang II binding affinity for the binding site suggesting that it is not a G protein‐coupled receptor. The semipurified, radio‐photolabeled binding site was applied for 2D gel electrophoresis. The isoelectric point of the binding site was ~ 7.0. Cyanogen bromide hydrolysis of the protein gave 2 bands: 25 & 12.5 kDa, suggesting that there are 3 or more Met residues in this protein. Mass‐spectroscopic analysis of these digests should provide a partial amino acid sequence, revealing whether this is a known protein with a novel functionality, or if it is a novel brain protein. Supported by the Peptide Radioiodination Service Center of the University of Mississippi.