Substrate Requirements for Angiotensin I Conversion In Vivo and In Vitro

Abstract

The substrate requirements for angiotensin I-converting enzyme were studied in vivo in the dog lung and in vitro in plasma, using angiotensin I (AI), 5- D -Ile-AI, 7- D -Pro-AI, and 8-D-Phe-AI which had been synthesized by the solid-phase technique. All peptides were inactive in the rabbit aortic strip preparation. None of the D-amino acid-substituted peptides gave a pressor response in the anesthetized dog; none showed significant immunologic cross-reactivity with anti-AI serum or antiangiotensin II serum. Each peptide was labeled with ¹²⁵ I, and the monoiodinated species was isolated. The iodinated peptides were incubated with diluted dog plasma or injected into the right ventricle of intact anesthetized dogs. In vitro, ¹²⁵ I-AI, ¹²⁵ I-5-D-IIe-AI, and ¹²⁵ I-7-D-Pro-AI were converted to angiotensin II (AII). 8-D-Phe-AI was not converted. In vivo, 15 seconds after injecting ¹²⁵ I-AI, ¹²⁵ I-5-D-IIe-AI, or ¹²⁵ I-7-D-Pro-AI into the right ventricle, ¹²⁵ I-AJI accounted for 70, 60, and 45%, respectively, of the radioactive material in aortic blood. These results and our previous observations on the importance of the C-terminal sequence of AI for conversion indicate that the enzymatic binding sites for AI-converting enzyme in vivo and in vitro extend from position 10 to position 8 but not to position 7. D-amino acid substitution at positions 7 and 5 abolishes biologic and immunologic activity without interfering with conversion.