A novel angiotensin analog with subnanomolar affinity for angiotensin-converting enzyme

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, th e binding characteristics of I-125-angiotensinogen 3-11(Lys(11)) were exami ned. Equilibrium saturation isotherms utilizing guinea pig lung membranes r evealed that I-125-angiotensinogen 3-11(Lys(11)) bound a single high-affini ty site in the presence of EDTA exhibiting a K sigma of 0.15 +/- 0.02 nM wi th a B-max = 4295 +/- 535 fmol/mg of protein. Competition studies revealed the following rank order of binding affinity: I-125-angiotensinogen 3-11(Ly s(11))>> bradykinin >> angiotensin I. Next, SDS-polyacrylamide gel electrop horesis analysis revealed that chemically cross-linked I-125-angiotensinoge n 3-11(Lys(11)) specifically bound a protein of M-r 173,000 that had the sa me 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 b inding 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 bindin g site for I-125-angiotensinogen 3-11(Lys(11)) and I-125-351A copurified. T hese experiments indicate that the novel angiotensin I analog, I-125-angiot ensinogen 3-11(Lys(11)) binds to ACE and suggest that there are critical bi nding sites outside the catalytic domains of ACE that determine binding spe cificity and affinity.