Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9) is a metallopeptidase that specifically removes N-terminal amino acids from peptides normally protected from attack by general aminopeptidases through the presence of proline in the penultimate N-terminal position. Since many biologically active peptides contain N-terminal Pro residues, AP-P can have a key role in initiating the metabolism and subsequent inactivation of such peptides. There are two forms of mammalian AP-P, an integral membrane form and a less abundant cytosolic enzyme. The membrane AP-P is a kininase, degrading the potent vasodilator bradykinin in the rat pulmonary vascular bed, the rat heart and in the human skin. It is also involved in collagen turnover by hydrolysing collagen derived peptides and may have a role in regulating peptides of the immune system. In contrast, the physiological roles of the mammalian cytosolic AP-P are unknown. The sequencing of the entire genome of Caenorhabditis elegans has identified several aminopeptidase genes, one of which ( W03G9.4 ) shows high homology to mammalian cytoplasmic AP-P. We have generated His-tagged AP-P through high-level expression in Escherichia coli . The purified enzyme hydrolyses the Arg-Pro, Lys-Pro and Ala-Pro bonds of bradykinin, the C. elegans
flp-9 gene product KPSFVRFamide and locustatachykinin (GPSGFYGVRamide) respectively. Using bradykinin as the substrate, we have shown that the C. elegans AP-P has many of the properties of mammalian AP-P. For example, it is inhibited by chelators of divalent metal ions, has a neutral pH optimum and is inhibited by the selective AP-P inhibitor, apstatin (IC 50 = 1 m M). Spatio/temporal expression pattern analysis using a W03G9.4 promoter:: GFP transgene showed expression to be predominantly in the most anterior cells of the intestine (Int 1) from the L2 stage through to the adult. This restricted expression in the intestine indicates a specific role for the enzyme in gut function. Interestingly, the highest levels of expression of mammalian cytoplasmic AP-P occur in the pancreas.