Characterization of a specific binding site for angiotensin II in chicken liver

We have characterized a specific binding site for angiotensin II (AngII) in chicken liver membranes. Pseudo-equilibrium studies at 22 degrees C for 30 min have shown that this binding site recognizes AngII with a high affinity (pK(D) of 8.13 +/- 0.21). The binding sites are saturable and relatively abundant (maximal binding capacity varies from 0.318 to 0.88 pmol/mg of protein). Nonequilibrium kinetic analyses at 22 degrees C revealed a calculated kinetic pK(D) of 8.77 +/- 0.20. The binding site is pharmacologically distinct from the classic AngII receptors AT(1) and AT(2). Competitive binding studies with chicken liver membranes demonstrated the following rank order of effectiveness: AngII (human; Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) > AngI (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu) > AngIII (Arg-Val-Tyr-Ile-His-Pro-Phe) > AngIV (Val-Tyr-Ile-His-Pro-Phe) > Ang(1-7) (Asp-Arg-Val-Tyr-Ile-His-Pro) > PD123319 (1-[4(dimethylamino)3-methylphenyl]methyl-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid) > DuP753 (2-n-butyl-4-chloro-5 hydroxymethyl-1-[(2'-1H-tetrazol-5-yl)biphenyl-4-yl)methyl]imidazole. This atypical AngII binding site (chicken AT) was sensitive to increasing concentrations of DTT and Mn2+. The structure-activity relationship on position 1 of AngII showed that the primary N-terminal amine was essential for binding affinity ([Asp(1)]AngII > [Suc(1)]AngII greater than or equal to [Sar(1)]AngII), but modifications of the side chain in position 1 had less influence on the affinity ([Gly(1)]AngII > [Cys(1)]AngII approximate to [aminoisobutyryl(1)]AngII approximate to [Ser(1)]AngII >>> [Sar(1)]AngII). The presence of substantial quantities of this binding site in chicken liver membranes suggests the possibility that the chicken AT may play an important, yet unrecognized, role in the renin-angiotensin system.