Aminopeptidase P: Immunoaffinity purification and molecular characterisation

Lloyd, Georgina S.; Habgood, Nicolette; Hooper, Nigel M.; Turner, Anthony J.

doi:10.1042/bst021236s

Cited by 2 publications

(2 citation statements)

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“…The choice of degenerate oligonucleotide primers was determined from partial peptide sequences of purified pig kidney AP-P obtained in our laboratory [22] and from the protein sequence reported by Vergas Romero et al [19]. The sequences of the sense primers were : F1, 5h-CARATGGAYTGYAAWTGGG-3h ; F2, 5h-GGIGAYGTIAARATHTGGAT-3h.…”

Section: Cloning Of Pig Ap-p Cdnamentioning

confidence: 99%

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Molecular cloning and expression in COS-1 cells of pig kidney aminopeptidase P

Hyde

Hooper

Turner

1996

Biochemical Journal

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Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9), a key enzyme in the metabolism of the vasodilator bradykinin, has been cloned from a pig kidney cortex cDNA library following the use of the PCR to identify sub-libraries enriched in AP-P clones. The complete primary sequence of the enzyme has been deduced from a full-length cDNA clone. This predicts a protein of 673 amino acids with a cleavable N-terminal signal sequence and six potential N-linked glycosylation sites. A stretch of mainly hydrophobic amino acids at the C-terminus is predicted to co-ordinate the attachment of a glycosyl-phosphatidylinositol (GPI) membrane anchor. Although AP-P is a zinc metallopeptidase, the predicted primary sequence does not contain any recognizable zinc-binding motif. Transient expression of AP-P cDNA in COS-1 cells resulted in enzymic activity characteristic of AP-P, namely apstatin- and EDTA-sensitive hydrolysis of bradykinin and Gly-Pro-Hyp. The expressed protein was recognized as a polypeptide of M(r)91,000 under reducing conditions, following immunoblotting of COS-1 membranes with a polyclonal antibody raised against purified pig kidney AP-P. The presence of a GPI anchor on expressed AP-P was established by demonstrating release of the enzyme from a membrane fraction following treatment with bacterial phosphatidylinositol-specific phospholipase C and its corresponding conversion from an amphipathic to a hydrophilic form, as assessed by phase separation in Triton X-114. Sequence comparisons confirm that AP-P is a member of the proline peptidase family of hydrolytic enzymes and is unrelated in sequence to other brush-border membrane peptidases.

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Section: Cloning Of Pig Ap-p Cdnamentioning

confidence: 99%

“…20 % breakdown of substrate was observed. to obtain N-terminal sequence data for purified pig kidney AP-P have been unsuccessful due to blockage of the N-terminus [5,22]. The amino acid sequence in Figure 1 also differs from that in [19] at one additional position : Cys&$!…”

Section: Figure 1 Nucleotide and Deduced Amino Acid Sequence Of Pig Kmentioning

confidence: 99%