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, the binding characteristics of (125)I-angiotensinogen 3-11(Lys(11)) were examined. Equilibrium saturation isotherms utilizing guinea pig lung membranes revealed that (125)I-angiotensinogen 3-11(Lys(11)) 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: (125)I-angiotensinogen 3-11(Lys(11)) >> bradykinin >> angiotensin I. Next, SDS-polyacrylamide gel electrophoresis analysis revealed that chemically cross-linked (125)I-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 (125)I-angiotensinogen 3-11(Lys(11)) and the ACE inhibitor, (125)I-351A, were also compared. These experiments demonstrated that the binding distributions of (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A are identical in the guinea pig lung and testes. Finally, the purification of ACE from guinea pig serum was monitored with (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A binding. These results demonstrated that the binding site for (125)I-angiotensinogen 3-11(Lys(11)) and (125)I-351A copurified. These experiments indicate that the novel angiotensin I analog, (125)I-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.
Krebs
L T
LT
Department of Veterinary and Comparative Anatomy, Washington State University, Pullman, Washington, USA.
Hanesworth
J M
JM
Sardinia
M F
MF
Speth
R C
RC
Wright
J W
JW
Harding
J W
JW
D000803:Angiotensin I / Q000031:analogs & derivatives* / Q000494:pharmacology; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology*; D000809:Angiotensins / Q000494:pharmacology*; D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive / Q000187:drug effects; D001920:Bradykinin / Q000494:pharmacology; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; D008168:Lung / Q000187:drug effects / Q000378:metabolism; D008297:Male; D007703:Peptidyl-Dipeptidase A / Q000097:blood / Q000378:metabolism*; D011485:Protein Binding
D000806:Angiotensin-Converting Enzyme Inhibitors; D000809:Angiotensins; D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D007457:Iodine Radioisotopes; C406524:angiotensinogen 3-11(Lys(11)); D000803:Angiotensin I; D007703:Peptidyl-Dipeptidase A; D001920:Bradykinin