Aminopeptidase N

Several enzymes, none of which are completely specific for the enkephalins, are known to cleave Leu- and Met-enkephalin at various peptide bonds. The main enzymes that degrade enkephalin are 2inc metaHopeptidases. The first enkephalin-degrading enzyme to be identified, an aminopeptidase which cleaves the amino terminal Tyr-Gly bond (179), has been shown to be aminopeptidase-N (APN) (180). It is a cytoplasmic enzyme which is uniformly distributed throughout the brain. The increased analgesic activity of synthetic enkephalins substituted by D-amino acids at position 2, eg,... 

Pasqualini R, Arap W, Koivunen E et al. Aminopeptidase N is a receptor for tumor-homing peptides and a target for inhibiting angiogenesis. Cancer Res 2000 60 722-727. 

Moffatt S, Wiehle S, Cristiano RJ (2005) Tumor-specific gene delivery mediated by a novel peptide-polyethylenimine-DNA polyplex targeting aminopeptidase N/CD13. Hum Gene Ther 16 57-67... 

Bestatin (ubenimex) is a potent inhibitor of aminopeptidase N and aminopeptidase B,89 which was isolated from a culture filtrate of Streptomyces olivoreticuli during the search for specific inhibitors of enzymes present on the membrane of eukaryotic cells.90 Inhibitors of aminopeptidase activity are associated with macrophage activation and differentiation, Bestatin has shown significant therapeutic effects in several clinical trials.91 In a multi-institutional study,92 patients with acute non-lymphocytic leukemia (ANLL) were randomized to receive either Bestatin or placebo orally after completion of induction and consolidation therapy, and concomitant with maintenance chemotherapy. Remission duration was prolonged in the Bestatin group, although this difference did... 

Aminopeptidase N. This enzyme works in a manner similar to carboxypeptidase Y, but cleaves amino acids from the N-terminus. It can be used in the same way as carboxypeptidase Y. 

Juillerat-Jeanneret L, Aubert JD, Leuenberger P (1997) Peptidases in human bronchoalveolar lining fluid, macrophages, and epithelial cells Dipeptidyl (amino)peptidase IV, aminopeptidase N, and dipeptidyl (carboxy)peptidase (angiotensin-converting enzyme). J Lab Clin Med 130(6) 603—614. 

X-ray crystallography of the 65-kDa form reveal a three-domain structure. The central domain varies among different strains and is probably involved in recognition and in binding to cell surface receptors.e h The toxin binds to a receptor, apparently an aminopeptidase N, after which the toxin is rapidly inserted into the membrane forming a 1- to 2-nm diameter pore. This leads to cell death.)... 

Cumis, F., Sacchi, A., Borgna, L., Magni, F., Gasparri, A. and Corti, A. (2000) Enhancement of tumor necrosis factor alpha antitumor immunotherapeutic properties by targeted delivery to aminopeptidase N (CD13). Nature Biotech., 18, 1185-1190. 

Also jV-acetylcysteine is capable of inhibiting aminopeptidase N [30]. Beside its low toxicity, this compound displays mucolytic properties that lead to a reduction of the diffusion barrier [31]. 

Bernkop-Schniirch, A., and M.K. Marschiitz. 1997. Development and in vivo evaluation of systems to protect peptide drugs from aminopeptidase N. Pharm Res 14 181. 

Bernkop-Schniirch, A., C. Paikl, and C. Valenta. 1997. Novel bioadhesive chitosan-EDTA conjugate protects leucine enkephalin from degradation by aminopeptidase N. Pharm Res 14 917. 

Bernkop-Schniirch, A., G. Walker, and H. Zarti. 2001. Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N. J Pharm Sci 90 1907. 

Valenta, C., et al. 2002. Evaluation of the inhibition effect of thiolated poly(acrylates) on vaginal membrane bound aminopeptidase N and release of the model drug LH-RH. J Pharm Pharmacol 54 603. 

Luciani, N., et al. 1998. Characterization of Glu350 as a critical residue involved in the N-terminal amine binding site of aminopeptidase N (EC 3.4.11.2) Insights into its mechanism of action. Biochemistry 37 686. 

Inoue, T., et al. 1994. Bestatin, a potent aminopeptidase-N inhibitor, inhibits in vitro decidualiza-tion of human endometrial stromal cells. J Clin Endocrinol Metab 79 171. 

Aminopeptidase N phosphinic acid dipeptide analogues (PADAs), 1,10-phenanthroline, puromycin Amastatin, amino acids, ACDs, bacitracin, bestatin, chitosan-EDTA conjugates, di- and tripeptides, EDTA, Na-glycocholate, PADAs, puromycin... 

There are at least three peptidases in the brush border of the small intestine Aminopeptidase A, which has an affinity for peptide-bound acid amino acids (11), aminopeptidase N, which has a broad specificity (12), and dipeptidylpeptidase IV, which releases dipeptides from the N-terminal end of peptides with a preference for X-PRO terminals (13). In Table III are shown the effect of a low concentration ofThe LMW fraction on the activity of these enzymes in extracts of hog intestine. Aminopeptidase N was found to be strongly inhibited by 0.25 mg/ml of the fraction. Aminopeptidase A and dipeptidylpeptidase IV were not inhibited. 

Since the glucose-lysine reaction mixture used in this study consisted of a number of different substances, it was of interest to study whether the observed inhibitory effect in vitro could be attributed to some specific compound(s). In order to obtain a separation, an aliquot of the LMW fraction, radiolabel led by [U- glucose added to the reactants, was applied on a Sephadex G-50 column and eluted with water. The UV absorbance was recorded and the eluate was collected in fractions. The degree of inhibition effected by small samples of equal volume from each fraction and exerted on carboxypeptidase A and purified aminopeptidase N was determined as well as the radioactivity... 

Figure 1. Separation of the low molecular weight (LMW) fraction on Sephadex G-50 superfine (2.6 X 90 cm, eluent water) (1). Key —, UV absorbance shaded area, relative distribution of a (,4C)-label on reactant glucose A, inhibition of aminopeptidase N and inhibition of carboxypeptidase A.

It is clear that the LMW fraction from the glucose-lysine reaction mixture affected, down to a concentration of at least 0.25 mg/ml, the rates of hydrolysis catalyzed by some, but not all, of the enzymes involved in protein digestion in vitro. Car-boxypeptidase A and aminopeptidase N were inhibited by a number of substances in the mixture, some being more effective than others. Efforts to characterize these compounds are in progress. 

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