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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Preview
Unable to display preview. Download preview PDF.
References
R. B. Harris and I. B. Wilson, J. Biol. Chem., 257: 811–815 (1982).
R. B. Harris and I. B. Wilson, J. Biol. Chem., 258: 1357–1362 (1983).
R. B. Harris and I. B. Wilson, Intl. J. Pep. Prot. Res., 20: 167–176 (1983).
D. H. Spackman, W. H. Stein, and S. Moore, Anal. Biochem., 30: 1190–1206 (1958).
P. Edman and A. Henschen, in: “Protein Sequence Determination,” S. B. Needleman, ed., Springer-Verlag, New York (1975).
M. T. Kimmel and T. H. Plummer, Jr., J. Biol. Chem., 247:7864–7869 (11972), Nature, New Biol., 238: 35–37 (1972).
D. Rasnick and J. C. Powers, Biochemistry, 17: 4363–4369 (1978).
R. A. Bradshaw, L. H. Ericsson, K. A. Walsh, and H. Neurath, Proc. Natl. Acad. Sci. USA, 63: 1389–1393 (1969).
K. Titani, M. A. Hermodson, L. H. Ericsson, K. A. Walsh, and H. Neurath, Nature, New Biol., 238: 35 – 37 (1972).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1986 Plenum Press, New York
About this chapter
Cite this chapter
Harris, R.B. (1986). Isolation and Sequencing of an Active-Site Peptide from Angiotensin I-Converting Enzyme. In: Greenbaum, L.M., Margolius, H.S. (eds) Kinins IV. Advances in Experimental Medicine and Biology, vol 198A. Springer, Boston, MA. https://doi.org/10.1007/978-1-4684-5143-6_68
Download citation
DOI: https://doi.org/10.1007/978-1-4684-5143-6_68
Publisher Name: Springer, Boston, MA
Print ISBN: 978-1-4684-5145-0
Online ISBN: 978-1-4684-5143-6
eBook Packages: Springer Book Archive