RT Journal Article
SR Electronic
T1 A Novel Angiotensin Analog with Subnanomolar Affinity for Angiotensin-Converting Enzyme
JF Journal of Pharmacology and Experimental Therapeutics
JO J Pharmacol Exp Ther
FD American Society for Pharmacology and Experimental Therapeutics
SP 260
OP 267
VO 293
IS 1
A1 Luke T. Krebs
A1 Jodie M. Hanesworth
A1 Michael F. Sardinia
A1 Robert C. Speth
A1 John W. Wright
A1 Joseph W. Harding
YR 2000
UL http://jpet.aspetjournals.org/content/293/1/260.abstract
AB 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 that125I-angiotensinogen 3–11(Lys11) bound a single high-affinity site in the presence of EDTA exhibiting aKd of 0.15 ± 0.02 nM with aBmax = 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 ofMr 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 for125I-angiotensinogen 3–11(Lys11) and125I-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. The American Society for Pharmacology and Experimental Therapeutics