Inhibitor profile of porcine aminopeptidase W.

Aminopeptidase W (APW; EC 3.4.1 I . 16) belongs to the family of mammalian cell-surface zinc metallopeptidases [ 13. These enzymes are involved in a number of different processes depending upon their location, metabolizing a range of different biologically active peptides including peptide hormones, neuropeptides and dietary peptides. Some of these cell-surface peptidases are proving to be important therapeutic targets in a variety of disease states, including heart disease, metastasis and inflammation. Some cell-surface peptidases have been shown to be identical to cluster differentiation (CD) antigens, eg endopeptidase-24.11 is identical to the common acute lymphoblastic leukaemia antigen (CALLA, CDlO) [2] and aminopeptidase N (APN; EC 3.4.11.2) is identical to CD13 [3]. In addition, APN has recently been shown to be a receptor for the human and porcine coronaviruses [4, 51. APW has been located to the microvilli of both the small intestine and the proximal convoluted tubules of the kidney, and has recently been identified in the peripheral nervous system [6, 71. APW releases N-terminal residues from short peptides with aromatic amino acids in the penultimate position [S]. Tryptophan is the most preferred penultimate amino acid. APW has an overlap of substrate and inhibitor specificities with APN and aminopeptidase A (APA; EC 3.4.1 1,7), this has made it difficult to determine the exact metabolic role of APW. In the quest for a specific inhibitor of APW, the effect of a range of metallopeptidase inhibitors on aminopeptidases W, N and A were compared. APA was assayed in porcine kidney microvillar membranes (5Opg) with aGlu-AMC (0.2mM) as substrate in 0.1M Tris/HCI, ImM CaC12, pH 7.4 at 37OC. The product AMC was detected flourimetrically at an excitation wavelength of 370nm and an emission wavelength of 442nm. APN (100ng) was assayed with Ala-AMC (0.2mM) as substrate in 0.1M TridHCI, pH 7.4 at 37OC. APW was assayed with Asp-Phew2 (ImM) as substrate in 0.1M Tris/HCl, pH 7.4 at 37OC. The product PheNH2 was separated from the substrate and was quantified by reverse phase HPLC as described for Gly-D-Phe [9]. Incubations were performed in triplicate with each concentration of inhibitor. There was an overlap in the inhibitory effects of known aminopeptidase inhibitors on aminopeptidases W, N and A. Amastatin inhibited aminopeptidases W, N and A with comparable potency, while probestin was a more potent inhibitor of APN (Table I ) . Actinonin was very selective and potent towards APN as compared with the other two aminopeptidases. Bestatin, which has been considered to be a potent inhibitor of APN, was found to be a more potent inhibitor of APW (Table I ) Thus the observed immunomodulating and antiturnour effects of bestatin could be due to its preferential inhibition of cell-surface APW. Amongst the sulphydryl, carboxyalkyl and phosphoryl inhibitors of angiotensin converting enzyme (EC 3.4.15.1) only the sulphydryl compounds, rentiapril, zofenoprilat and YS980 displayed significant inhibitory action towards APW (Table 1).