A SPECIFIC BINDING-SITE RECOGNIZING A FRAGMENT OF ANGIOTENSIN-II IN BOVINE ADRENAL-CORTEX MEMBRANES

We have characterized a specific binding site for angiotensin IV in bovine adrenal cortex membranes. Pseudo-equilibrium studies at 37 degrees C for 2 h have shown that this binding site recognizes angiotensin IV with a high affinity (K-d = 0.24, +/-0.03 nM). The binding site is saturable and relatively abundant (maximal binding capacity around 0.5 pmol/mg protein). Non-equilibrium kinetic analyses at 37 degrees C revealed a calculated kinetic K-d of 47 pM. The binding site is pharmacologically distinct from the classic angiotensin receptors AT(2) or AT(2). Competitive binding studies with bovine adrenal cortex membranes demonstrated the following rank order of effectiveness: angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) = angiotensin II-(3-7) (Val-Tyr-Ile-His-Pro). angiotensin III (Arg-Val-Tyr-Ile-His-Pro-Phe) greater than or equal to angiotensin II-(4-7) (Tyr-Iie-His-Pro) > angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe)> angiotensin II-(1-6) (Asp-Arg-Val-Tyr-Ile-His) > angiotensin II-(4-8) (Tyr-Ile-His-Pro-Phe) > > > angiotensin II-(3-6) (Val-Tyr-Ile-His), angiotensin II-(4-6) (Tyr-Ile-His), L-158,809 (5,7-dimethyl-2-ethyl-3-[(2'-(1 H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl)methyl]-3H-imidazo[4,5- beta]pyridine H2O) and PD 123319 (1-[4-(dimethylamino)3-methylphenyl]methyl-5- (diphenylacetyl)4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine- 6-carboxylic acid). The divalent cations Mg2+ and Ca2+ were shown to diminish the binding of I-125-angiotensin IV to bovine adrenal cortex membranes. The presence of a high concentration of this binding site in the adrenal cortex suggests the possibility that it may play an important, yet unrecognized role, in the renin-angiotensin system.