Vol. 293, Issue 1, 260-267, April 2000

A Novel Angiotensin Analog with Subnanomolar Affinity for Angiotensin-Converting Enzyme¹

Luke T. Krebs, Jodie M. Hanesworth, Michael F. Sardinia, Robert C. Speth, John W. Wright and Joseph W. Harding

Department of Veterinary and Comparative Anatomy, Pharmacology, and Physiology, Washington State University, Pullman, Washington

This study demonstrates that a novel angiotensin I analog, angiotensinogen 3-11(Lys¹¹), possesses a high affinity for angiotensin-converting enzyme (ACE), which is substantially greater than the endogenous substrates. This assessment is based on data derived from a variety of techniques. First, the binding characteristics of ¹²⁵I-angiotensinogen 3-11(Lys¹¹) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that ¹²⁵I-angiotensinogen 3-11(Lys¹¹) bound a single high-affinity site in the presence of EDTA exhibiting a K_d of 0.15 ± 0.02 nM with a B_max = 4295 ± 535 fmol/mg of protein. Competition studies revealed the following rank order of binding affinity: ¹²⁵I-angiotensinogen 3-11(Lys¹¹) bradykinin angiotensin I. Next, SDS-polyacrylamide gel electrophoresis analysis revealed that chemically cross-linked ¹²⁵I-angiotensinogen 3-11(Lys¹¹) specifically bound a protein of M_r 173,000 that had the same molecular weight as ACE. Utilizing in vitro autoradiography, the binding distributions of ¹²⁵I-angiotensinogen 3-11(Lys¹¹) and the ACE inhibitor, ¹²⁵I-351A, were also compared. These experiments demonstrated that the binding distributions of ¹²⁵I-angiotensinogen 3-11(Lys¹¹) and ¹²⁵I-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with ¹²⁵I-angiotensinogen 3-11(Lys¹¹) and ¹²⁵I-351A binding. These results demonstrated that the binding site for ¹²⁵I-angiotensinogen 3-11(Lys¹¹) and ¹²⁵I-351A copurified. These experiments indicate that the novel angiotensin I analog, ¹²⁵I-angiotensinogen 3-11(Lys¹¹) binds to ACE and suggest that there are critical binding sites outside the catalytic domains of ACE that determine binding specificity and affinity.