This study demonstrates that a novel angiotensin I analog, angiotensinogen 3-11(Lys(11)), possesses a high affinity for angiotensin-converting enzyme (ACE), which is substantially greater than the endogenous substrate...
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This study demonstrates that a novel angiotensin I analog, angiotensinogen 3-11(Lys(11)), 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 I-125-angiotensinogen 3-11(Lys(11)) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that I-125-angiotensinogen 3-11(Lys(11)) bound a single high-affinity site in the presence of EDTA exhibiting a K sigma 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: I-125-angiotensinogen 3-11(Lys(11))>> bradykinin >> angiotensin I. Next, SDS-polyacrylamide gel electrophoresis analysis revealed that chemically cross-linked I-125-angiotensinogen 3-11(Lys(11)) 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 I-125-angiolensinogen 3-11(Lys(11)) and the ACE inhibitor, I-125-351A, were also compared. These experiments demonstrated that the binding distributions of I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A binding. These results demonstrated that the binding site for I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A copurified. These experiments indicate that the novel angiotensin I analog, I-125-angiotensinogen 3-11(Lys(11)) binds to ACE and suggest that there are critical binding sites outside the catalytic domains of ACE that determine binding specificity and affinity.
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