Photoaffinity labeling of membrane-bound porcine aminopeptidase N

To investigate the possible role of aminopeptidase N (alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2) in the transport of amino acids from oligopeptides, the modified amino acids Phe(N3) and Phe(N3, I) and the tetrapeptides Phe(N3) or Phe(N3, I)-L-or-DAla-Gly-Gly have been synthesized. The azido-amino acids were radioactively labeled by tritium or 125I before their coupling with the tripeptides. Their utilization as photoaffinity labels for aminopeptidase N has been studied. The modification imposed at the N-terminal residue of the tetrapeptides has not impaired their hydrolysis by porcine aminopeptidase N (same kinetic parameters as unmodified peptides). In addition, evidence is presented for a specific and reversible interaction in the dark of the azido-derivatives at the substrate recognition site of the enzyme. Upon photolysis, irreversible inactivation of aminopeptidase N and covalent attachment of Phe(N3, I) have been demonstrated. Soluble and membrane-bound aminopeptidases are both labeled to the same extent indicating that the free azido-amino acid preferentially reacts with the external part of the enzyme. Although the linkage of the azido-derivative is not strictly restricted to the region of the active site, the values obtained strongly suggest that 1 mol probe has been covalently attached per mol monomer of inhibited aminopeptidase.