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PubMed1976

Nature of the multiple forms of alanine aminopeptidase.

Alanine aminopeptidase (particle-bound aminopeptidase, EC 3.4.11.2) isolated in a highly purified state from human liver, kidney, and pancreas were investigated with regard to their different electrophoretic mobilities. While the molecular weights are identical, different isoelectric points were found. The results of carbohydrate analysis point to N-acetylneuraminic acid as the main factor that causes the differences in electrophoretic behaviour of the alanine aminopeptidases investigated.

Amino Sugars↗

PubMed2003

Aminopeptidase inhibitor bestatin stimulates microvascular endothelial cell invasion in a fibrin matrix.

The aminopeptidase inhibitor bestatin has been shown to have anti-angiogenic effects in a number of model systems. These effects are thought to result from inhibition of CD13 activity. Because tumor angiogenesis can evolve in a fibrin-rich stroma matrix we have studied for the first time the effects of bestatin on microvascular endothelial capillary-like tube formation in a fibrin matrix. Bestatin enhanced the formation of capillary-like tubes dose-dependently. Its effects were apparent at 8 micro M; the increase was 3.7-fold at 125 micro M; while high concentrations (>250 micro M), that were shown to have anti-angiogenic effects in other systems, caused extensive matrix degradation. Specific CD13-blocking antibodies WM15 and MY-7, and the aminopeptidase inhibitors amastatin and actinonin also enhanced capillary-like tube formation (maximally 1.5-fold), but these effects did not reach statistical significance. The effect of bestatin was not due to a change in uPAR availability because the relative involvement of the u-PA/u-PAR activity was not altered by bestatin. In view of the present findings we hypothesize that aminopeptidases other than CD13 predominantly contribute to the observed pro-angiogenic effect of bestatin in a fibrin matrix. The identification of this novel effect of bestatin is important in the light of the proposed use of bestatin as anti-angiogenic and/or anti-tumor agent.

Aminopeptidases↗

PubMed2004

Miniglucagon (MG)-generating endopeptidase, which processes glucagon into MG, is composed of N-arginine dibasic convertase and aminopeptidase B.

Miniglucagon (MG), the C-terminal glucagon fragment, processed from glucagon by the MG-generating endopeptidase (MGE) at the Arg17-Arg18 dibasic site, displays biological effects opposite to that of the mother-hormone. This secondary processing occurs in the glucagon- and MG-producing alpha-cells of the islets of Langerhans and from circulating glucagon. We first characterized the enzymatic activities of MGE in culture media from glucagon and MG-secreting alphaTC1.6 cells as made of a metalloendoprotease and an aminopeptidase. We observed that glucagon is a substrate for N-arginine dibasic convertase (NRDc), a metalloendoprotease, and that aminopeptidase B cleaves in vitro the intermediate cleavage products sequentially, releasing mature MG. Furthermore, immunodepletion of either enzyme resulted in the disappearance of the majority of MGE activity from the culture medium. We found RNAs and proteins corresponding to both enzymes in different cell lines containing a MGE activity (mouse alphaTC1.6 cells, rat hepatic FaO, and rat pituitary GH4C1). Using confocal microscopy, we observed a granular immunostaining of both enzymes in the alphaTC1.6 and native rat alpha-cells from islets of Langerhans. By immunogold electron microscopy, both enzymes were found in the mature secretory granules of alpha-cells, close to their substrate (glucagon) and their product (MG). Finally, we found NRDc only in the fractions from perfused pancreas that contain glucagon and MG after stimulation by hypoglycemia. We conclude that MGE is composed of NRDc and aminopeptidase B acting sequentially, providing a molecular basis for this uncommon regulatory process, which should be now addressed in both physiological and pathophysiological situations.

Aminopeptidases↗

PubMed1994

Bestatin, a potent aminopeptidase-N inhibitor, inhibits in vitro decidualization of human endometrial stromal cells.

We have reported that human endometrial stromal cells (ESC) express a cluster of differentiation-13 antigen/aminopeptidase-N, and the expression of this peptidase antigen was shown to increase with the decidualization of ESC. To clarify the role of this peptidase in human endometrium, the effect of bestatin ([(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-(S)-leucine), an inhibitor of aminopeptidase-N, on the decidualization of ESC in vitro was examined. Purified human ESC were cultured for 12 days in the presence of 10(-6) mol/L progesterone with or without bestatin. Decidualization was assessed by PRL production and morphological transformation. The effects of a stereoisomer of bestatin and of pepstatin were similarly examined using the same culture system. Bestatin inhibited progesterone-induced PRL production in a dose-dependent manner, with no effect on cell number or viability, whereas neither its stereoisomer nor pepstatin inhibited aminopeptidase activity or PRL production. The morphological transformation of ESC was also inhibited by bestatin, but not by its stereoisomer or pepstatin. These findings demonstrate that the inhibition of aminopeptidase-N activity blocks the in vitro decidualization of ESC and suggest an important role for this peptidase in the functional differentiation of human ESC.

Adult↗

PubMed2004

Alzheimer's, angiotensin IV and an aminopeptidase.

The angiotensin AT(4) receptor was originally defined as the specific, high affinity binding site for the hexapeptide angiotensin IV (Ang IV). Subsequently, the peptide LVV-hemorphin 7 was also demonstrated to be a bioactive ligand of the AT(4) receptor. Central administration of Ang IV or LVV-hemorphin 7 (LVV-H7) markedly enhances learning and memory in normal rodents and reverse memory deficits observed in animal models of amnesia. The high affinity binding site has a broad distribution in the brain including areas such as the hippocampus that are involved in memory processing. The high affinity Ang IV binding site (AT(4) receptor) has been identified as the transmembrane enzyme, insulin-regulated membrane aminopeptidase (IRAP). Insulin-regulated aminopeptidase is a type II integral membrane spanning protein belonging to the M1 family of aminopeptidases and in insulin-responsive cells colocalizes with GLUT4 in specific intra-cellular vesicles. Both Ang IV and LVV-H7 are competitive inhibitors of IRAP catalytic activity and are not substrates of the enzyme.

Alzheimer Disease↗

PubMed2006

A derivative of aminopeptidase inhibitor (BE15) has a dual inhibitory effect of invasion and motility on tumor and endothelial cells.

Bestatin is an inhibitor of aminopeptidase N (APN)/CD13 and aminopeptidase B. In our previous report, bestatin inhibited the tumor cell invasion and the angiogenesis induced by the inoculation of B16-BL6 melanoma cells into mice and capillary formation on human umbilical vein endothelial cells (HUVECs) in vitro. The results show that the enzymatic activity of APN is deeply involved in tumor invasion and angiogenesis. We investigated the effect of three bestatin derivatives on A375 human melanoma cells and in vitro. All the derivatives inhibited the activity of APN, but BE15 was most effective and controlled the migration of A375 cells and HUVECs and capillary formation of HUVECs. Furthermore, the bestatin derivatives had an inhibitory effect not only on aminopeptidase activity but also on cell motility. Compared with bestatin and the other derivatives, BE15 had a marked inhibitory effect on the formation of capillary structure by HUVECs in vitro. These results suggest that new anti-metastatic and anti-angiogenic agents, which have a dual inhibitory effect on the degradation of the extra cellular matrix and cell motility, may be developed from bestatin.

Aminopeptidases↗

PubMed1997

Purification and properties of an aminopeptidase from a protamine-degrading marine bacterium.

A protamine-degrading marine bacterium was isolated from marine soil and identified as Aeromonas salmonicida subsp. based on its taxonomical characteristics. An alanine-specific aminopeptidase, called aminopeptidase K, from an extract of the strain was purified and characterized. The aminopeptidase K was purified about 80-fold by fractionation with ammonium sulfate and column chromatography on QA-52 cellulose, Phenyl Superose and Superose 12. The purified enzyme is composed of 6 subunits of 86 kDa with a molecular mass of 520 kDa according to gel filtration and SDS-PAGE. The N-terminal sequence of the enzyme was H.Gly-Gln-Gln-Pro-Gln-Ile-Lys-Try-Tyr-His-Asp-Tyr-Asp-Ala-Pro-Asp-Tyr-Ty r- Ile-Thr-. It is inhibited by monoiodoacetate, N-ethylmaleimide, and puromycin. The Michaelis constant (K(m)) and the maximal rate of hydrolysis (Vmax) were, respectively, 0.28 mM and 49.4 mumol/min/mg for the L-Ala-beta-naphthylamide substrate. The optimum pH and optimum temperature were 6.5 and 45 degrees C, respectively. The purified enzyme was highly specific to L-Ala-beta-naphthylamide.

Aeromonas↗

PubMed2000

Purification and some properties of an aminopeptidase from the seeds of Cannabis sativa.

An aminopeptidase (HSA) with a molecular mass of 78 kDa was purified from hemp (Cannabis sativa) seeds. The activity was inhibited by monoiodeacetic acid, p-chloromercuri-phenylsulfonic acid, and Zn2+ ion. The specificity of HSA was similar to that of a leucyl aminopeptidase [EC 3.4.11.1] from mammalian cytosol. However, other enzyme properties were different from these of leucyl aminopeptidase.

Aminopeptidases↗

PubMed2004

Cathepsin L and Arg/Lys aminopeptidase: a distinct prohormone processing pathway for the biosynthesis of peptide neurotransmitters and hormones.

Peptide neurotransmitters and hormones are synthesized as protein precursors that require proteolytic processing to generate smaller, biologically active peptides that are secreted to mediate neurotransmission and hormone actions. Neuropeptides within their precursors are typically flanked by pairs of basic residues, as well as by monobasic residues. In this review, evidence for secretory vesicle cathepsin L and Arg/Lys aminopeptidase as a distinct proteolytic pathway for processing the prohormone proenkephalin is presented. Cleavage of prohormone processing sites by secretory vesicle cathepsin L occurs at the NH2-terminal side of dibasic residues, as well as between the dibasic residues, resulting in peptide intermediates with Arg or Lys extensions at their NH2-termini. A subsequent Arg/Lys aminopeptidase step is then required to remove NH2-terminal basic residues to generate the final enkephalin neuropeptide. The cathepsin L and Arg/Lys aminopeptidase prohormone processing pathway is distinct from the proteolytic pathway mediated by the subtilisin-like prohormone convertases 1/3 and 2 (PC1/3 and PC2) with carboxypeptidase E/H. Differences in specific cleavage sites at paired basic residue sites distinguish these two pathways. These two proteolytic pathways demonstrate the increasing complexity of regulatory mechanisms for the production of peptide neurotransmitters and hormones.

Aminopeptidases↗

PubMed1980

Endopeptidase of the brush border membrane of rat enterocyte. Separation from aminopeptidase and partial characterization.

The brush border of the enterocytes of the rat was isolated by the method of differential centrifugation with CaCl2 according to Schmitz. This material was solubilized with papain, trypsin and Triton X-100. The greatest amount of membrane enzymes was released to the supernatant (105 000 X g) with the use of Triton X-100. The tritonized supernatant was treated in the next step by papain, bromelain, ficin and trypsin (individually or in combinations). After simultaneous proteolysis with papain and bromelain a partial separation of the aminopeptidase from the endopeptidase by Sephadex G-200 chromatography was observed. These two enzyme activities were distinctly separated by isoelectric focusing at pH 4--6. Two enzymatically active bands (RF 0.13 and 0.24) in the aminopeptidase fraction and one single active band (RF 0.16) in the endopeptidase fraction using polyacrylamide gel electrophoresis were found. Co-migrating proteins to all of these activities were detected. Endopeptidase activity splits 3-carboxypropionyltrialanin-4-nitroanilide (SucAla3NAp) in the position P2-P1. Liberated aminoacyl-NAP may be further split to generate chromogenic 4-nitroaniline through aminopeptidase activity. Endopeptidase of the brush border of the rat enterocytes is characterized by the following properties: 1) molecular mass 130000 +/- 15 000 dalton; 2) Km value (substrate: SucAla3NAp) 1.1 X 10(-3) M; 3) pI 5.23; 4) ph optimum 8.5; 5) 50% activity remains after 15 min of preincubation at 50 degrees C; 6) activity is strongly inhibited by EDTA, p-chloromercuribenzoate, Mn2 and Co2.

Aminopeptidases↗

PubMed1992

Basic amino acids preferring broad specificity aminopeptidase from human erythrocytes.

An aminopeptidase hydrolyzing 2-naphthylamides of Lys, Arg, Leu, Met, Phe and Tyr, as well as different di- to tridecapeptides, was purified from the cytosol of human erythrocytes. The enzyme showed preference for Lys and Arg at N-terminus, as proline and D-amino acids were nonpermissive at P1' site. Higher affinity for oligopeptides than for aminoacyl naphthylamides was observed. Among the substrates were Lys-bradykinin, angiotensin III, thymopentin and enkephalins. Aminopeptidase was shown to be a monomeric protein of M(r) approximately 110000 and of pI approximately 4.8, activated by Co2+ and inhibited by EDTA, pHMB, amastatin, bestatin and puromycin. The isolated enzyme could be classified as cytosolic, Lys(Arg) preferring, broad specificity aminopeptidase.

Amino Acid Sequence↗

PubMed2005

The significance of cathepsins, thrombin and aminopeptidase in diffuse interstitial lung diseases.

To determine the significance of proteases in interstitial lung diseases, we examined the activity of cathepsins, thrombin, and aminopeptidase in bronchoalveolar lavage (BAL) fluid from patients with these disorders. Significantly increased activities of cathepsin H and aminopeptidase were detected in BAL fluid from patients with idiopathic pulmonary fibrosis (IPF), cryptogenic organizing pneumonia (COP), chronic eosinophilic pneumonia (CEP) and hypersensitivity pneumonitis (HP). Significantly higher activity of cathepsin B was found in BAL fluid from patients with CEP. The activity of thrombin was significantly higher in patients with IPF and CEP. In patients with IPF, there were significant correlations between neutrophil number and the activity of cathepsin B, cathepsin H or aminopeptidase. In patients with COP and HP, the activity of the proteases was significantly higher in patients with higher number of lymphocytes than in those with lower number of lymphocytes. The present study suggests that the activity of the proteases is a useful marker in activity of the interstitial lung diseases, and may have a role in the pathogenesis of these disorders.

Adult↗

PubMed2003

The insulin-regulated aminopeptidase: a companion and regulator of GLUT4.

The insulin-regulated membrane aminopeptidase (IRAP) was originally identified in fat and muscle cells as a major protein in intracellular vesicles that also harbor the insulin-responsive glucose transporter GLUT4. IRAP, like GLUT4, predominantly localizes to these intracellular vesicles under basal conditions. In response to insulin IRAP, like GLUT4, translocates to the plasma membrane. Purification and cloning of IRAP revealed that it was a novel member of the family of zinc-dependent membrane aminopeptidases. Upon the cloning of the human placental oxytocinase (P-LAP) it was discovered that IRAP and P-LAP were the rat and human homologues of the same protein. The expression of IRAP/P-LAP is not limited to fat and muscle cells, and the subcellular distribution of IRAP/P-LAP is regulated by different peptide hormones and exercise. IRAP/P-LAP cleaves several peptide hormones in vitro. In insulin- and oxytocin-treated cells, concomitant with the appearance of IRAP/P-LAP at the cell surface, aminopeptidase activity toward extracellular substrates increases. A physiological function for IRAP/P-LAP may thus be the processing of circulating peptide hormones. These extracellular substrates, however, would be processed efficiently only when IRAP/P-LAP gets access to them after translocation to the cell surface upon stimulation of cells with insulin or other factors. The in vivo substrates for IRAP/P-LAP remain to be determined. The initial characterization of mice in which IRAP/P-LAP was deleted (IRAP -/- mice) revealed that GLUT4 protein levels were dramatically decreased in all fat and muscle tissues. This finding suggests a function for IRAP/P-LAP in the regulation of GLUT4 levels. Further characterization of the IRAP -/- mice is required to elucidate the role IRAP/P-LAP may play in the control of peptide hormone metabolism.

Animals↗

PubMed1989

Novel inhibitors of enkephalin-degrading enzymes. II: N5'-substituted-4-thioxohydantoic acids as aminopeptidase inhibitors.

Some 2-substituted-(2'-aminophenyl)-4-thioxohydantoic acids (o-amino PTC-amino acids) have antinociceptive activity when administered (icv) alone (IC50 = 0.04-0.87 microM/animal) and show a striking prolongation of the antinociceptive action of (D-Ala-2 D-Leu5)-enkephalin (DADL) in combination. The effects are thought to be mediated via opioid receptors since they are naloxone-reversible. Although inhibitors of the enkephalin degrading puromycin-insensitive, bestatin-sensitive aminopeptidase (possibly aminopeptidase M) their action is weak (IC50 = 32 microM leucine, 536 microM, glycine) and they might be considered to have a direct antinociceptive effect on opioid receptors. The titled compounds constitute novel 'lead' compounds for the development of potent aminopeptidase M inhibitors.

Aminopeptidases↗

PubMed2005

Leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for MHC class I antigen presentation.

To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Leucine aminopeptidase (LAP) is an IFN-inducible cytosolic aminopeptidase that can trim precursor peptides to mature epitopes and has been thought to play an important role in Ag presentation. To examine the role of LAP in generating MHC class I peptides in vivo, we generated LAP-deficient mice and LAP-deficient cell lines. These mutant mice and cells are viable and grow normally. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full-length Ag constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. These data demonstrate that LAP is not an essential enzyme for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases.

Amino Acid Sequence↗

PubMed1985

Bestatin, a microbial aminopeptidase inhibitor, inhibits DNA synthesis induced by insulin or epidermal growth factor in primary cultured rat hepatocytes.

Bestatin, a microbial aminopeptidase inhibitor, inhibited insulin- or epidermal growth factor-induced DNA synthesis and cell proliferation in primary cultured hepatocytes of rats. The aminopeptidase inhibitor also affected the growth of FM3A or LOBN cells of mice, when it is included in the culture media at the concentration of 0.5 mg/ml. These results suggest the important role of the bestatin-sensitive aminopeptidase(s) in the process of DNA synthesis and proliferation of animal cells.

Aminopeptidases↗

PubMed2000

Function of the N-terminal propeptide of an aminopeptidase from Vibrio proteolyticus.

An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli. Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro. Using L-Leu-p-nitroanilide as the substrate, the kinetic parameters (k(cat) and K(m)) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.

Aminopeptidases↗

PubMed2002

Ability of Pseudomonas sp. to synthesize aminopeptidases in the presence of various carbon and nitrogen sources.

A soil strain of Pseudomonas sp. is able to synthesize at least two aminopeptidases exhibiting high activity in the presence of Phe-beta-NA and Ala-beta-NA as substrates. Irrespective of the used substrate, total activity of studied enzymes was strongly related to concentrations of organic components (peptone, glutamic acid, glucose) in mineral media and was the higher, the higher the concentration. Tendency of changes in total activity was similar for alanyl- and phenylalanylaminopeptidase though their response to different concentrations of organic components was different. Specific activity measured in the presence of Phe-beta-NA and Ala-beta-NA as the substrates was not strictly dependent on increasing concentrations of organic components in the media. The highest specific activity of aminopeptidase was obtained in the presence of Phe-beta-NA as a substrate on the fifth day of culture in medium containing 1% glucose. The obtained results seem to indicate the inductive character of the studied aminopeptidases. On the other hand, however, they do not exclude other regulatory mechanisms of their synthesis, including catabolic repression.

Aminopeptidases↗