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Kinins IV pp 513-521 | Cite as

Isolation and Sequencing of an Active-Site Peptide from Angiotensin I-Converting Enzyme

  • Robert B. Harris
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 198A)

Summary

A glutamic acid residue at the active-site of bovine lung antiotensin I-converting enzyme was esterified with p-[N,N-bis-(chloroethyl)amino]phenyl- butyryl-L-[U-14C]-Proline(chlorambucyl-L-[U-l4C]-L-Proline), an affinity label for this enzyme.2 The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase HPLC contained the bound radiolabel. This active-site peptide (Mr ~ 16,000) was digested with trypsin, and the labeled peptide (T-2) was further degraded with thermolysin. The enzyme digest peptides were also resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained after thermolysin digestion (Th-1, Mr 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu. The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of PTH- Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U−14C]-Proline which confirms our earlier findings. The sequence that we determined is homologous in five residues with the corresponding sequences of carboxypeptidase A and B, two other mammalian zinc-proteases. There is little sequence homology with thermolysin, a bacterial zinc-protease that also contains an essential active-site glutamic acid residue.

Keywords

Cyanogen Bromide Cleavage Peptide Glutamic Acid Residue Bovine Lung Affinity Label 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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Copyright information

© Plenum Press, New York 1986

Authors and Affiliations

  • Robert B. Harris
    • 1
  1. 1.Department of ChemistryUniversity of ColoradoBoulderUSA

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