ELUCIDATION OF A SPECIFIC BINDING-SITE FOR ANGIOTENSIN-II(3-8), ANGIOTENSIN-IV, IN MAMMALIAN HEART MEMBRANES

Data are presented describing a new angiotensin binding site in rabbit and guinea pig heart, distinct from AT1 and AT2, that demonstrates hi gh specificity and affinity for the hexapeptide fragment angiotensin I I(3-8), which will be referred to here as angiotensin IV (AIV). Equili brium binding in rabbit heart membranes was achieved in 2 hr at 37-deg rees-C and produced a calculated kinetic K(D) Of.1 74 +/- .018 nM. Sat uration equilibrium binding data for rabbit and guinea pig heart were best fit to a one-site model with Hill coefficients near unity. Guinea pig membranes exhibited a K(D) = 1.33 +/- .02 nM and a B(max) = 144 /- 19 fmol/mg protein, and rabbit heart membranes had a K(D) = 1.70 +/ - .50 nM and a B(max) = 731 +/- 163 fmol/mg protein. The binding site showed a high specificity for AIV, although it exhibited low affinity for angiotensin II, angiotensin III, Sar1,Ile8-angiotensin II, DuP 753 , CGP42112A and PD123177. A large number of nonangiotensin-related pep tides were unable to compete effectively for I-125-AIV binding. Deleti ons made from the C-terminal end of AIV caused a decrease in affinity: AIV > AII(3-7) much-greater-than-or-equal-to AII(3-6) much-greater-th an-or-equal-to AII(3-5). Extension of the C-terminal end of AIV corres ponding to the amino acids of human angiotensinogen caused little chan ge in affinity. GTP(gamma)S had no effect on binding, suggesting non-G protein linkage. Binding was widely distributed throughout the heart; it was observed on cardiocytes and blood vessels as well as in the ep icardium and the endocardium.