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PubMed1980

Effect of a proteinase inhibitor, aprotinin, on brush border membrane associated aminopeptidase of human kidney cortex.

In order to study the influence of the basic proteinase inhibitor aprotinin (Trasylol) on renal tubular alanine-aminopeptidase (E.C.3.6.11-), a brush border fraction was prepared from human kidney cortex by differential centrifugation. Incubation of brush border fragments, rich in membrane bound alanine-aminopeptidase, resulted in an activation of the enzyme. However, aminopeptidase, which was cleaved off from the membranes by limited proteolysis and subsequently purified by Concanavalin A-affinity chromatography exhibited activation by low but inhibition by higher concentration of aprotinin. Thus inhibition was associated with presence of the solubilized form of the brush border enzyme. On the other hand, as was shown by quantitative electroimmunoassay, formation of antigen-antibody complexes between soluble renal aminopeptidase and its specific rabbit antibody was not changed by the basic polypeptide.

Aminopeptidases↗

PubMed1982

Localization by immunofluorescence and histochemical labeling of aminopeptidase N in relation to its biosynthesis in rabbit and pig enterocytes.

Antibodies raised against highly purified rabbit intestinal brush border aminopeptidase N were found in certain rabbits or pigs to crossreact with human blood-grouplike substances present in goblet cells and at the surface of the basolateral membrane of enterocytes. To obtain antibodies strictly specific for aminopeptidase, depletion of antibodies with a water-insoluble, mucus-rich fraction or human A erythrocytes was necessary. By using ultrathin frozen sections of rabbit and pig jejunum mucosa, both the brush border and a structure at the upper pole of the nucleus were labeled by the specific antiaminopeptidase antibodies. This structure, tentatively identified as the Golgi apparatus in which aminopeptidase is glycosylated, escaped detection by histochemistry, thus suggesting that the enzyme is not yet active at this stage of its intracellular processing. By contrast, histochemistry revealed active aminopeptidase molecules in another structure located under the terminal web and in the upper part of the lateral membrane.

Aminopeptidases↗

PubMedDate not supplied

[Affinity chromatography of aminopeptidases].

The recent data are generalized concerning a series of synthetic oligopeptides which are competitive inhibitors of aminopeptidases of animal, plant and microbic origin. A method for biospecific chromatography of these enzymes is developed, using as ligands such inhibitors as diazo derivatives of p-aminophenyl-, chloromethyl- and methylketones of L-amino acids and peptides, amino acids, aliphatic acid amides. It is established that the most effective inhibitors of aminopeptidases contain L-amino group in the uncharged form in the N-end position, hydrophobic lateral chain of L-configuration and a carbonyl group analogous to position of these groups in the substrate. Methods for synthesis of certain peptides are developed with respect to the above requirements. It is shown that peptides with a space-inaccessible peptide link and antibiotics are often used as ligands for affinity chromatography of aminopeptidases. At present a nonspecific (ion-exchange, hydrophobic) interaction of sorbent and aminopeptidases is observed, which necessitates to increase the specificity at the stage of the enzyme desorption in the further studies.

Aeromonas↗

PubMed1993

Isolation and characterization of AAP1. A gene encoding an alanine/arginine aminopeptidase in yeast.

The yeast AAP1 gene, encoding a putative amino-peptidase, was isolated based on its ability to suppress the temperature-sensitive growth on nonfermentable carbon sources of spr5, a stationary phase regulatory mutant. AAP1 was physically mapped to chromosome VIII between PUT2 and CUP1. Sequence analysis of the AAP1 gene showed a 1581-nucleotide open reading frame capable of encoding a 59-kilodalton protein. The protein encoded by this open reading frame exhibits approximately 40% sequence identity to human, rat, and mouse aminopeptidases. In limited regions, sequence identity between Aap1 and the mammalian aminopeptidases ranges from 53% to 93%. Insertional inactivation of the AAP1 gene resulted in a decrease in glycogen accumulation and the loss of the major band of arginine/alanine aminopeptidase activity. Strains carrying the AAP1 gene on a high copy plasmid show an increase in the major arginine/alanine aminopeptidase activity, a dramatic increase in glycogen accumulation, and an increase in transcription from a vector carrying lacZ fused to the promoter of a gene (SSA3) expressed during post-diauxic and stationary phases of the culture cycle. We conclude that although the AAP1 gene is not essential for viability, the Aap1 protein positively affects glycogen accumulation in yeast.

Amino Acid Sequence↗

PubMed1995

Effect of a new aminopeptidase P inhibitor, apstatin, on bradykinin degradation in the rat lung.

Bradykinin (Bk), a potent vasoactive and cardioprotective peptide hormone, is almost completely inactivated during a single circulation through the rat lung. It has been hypothesized that membrane-bound aminopeptidase P, which can hydrolyze the Arg1-Pro2 bond of Bk, and angiotensin-converting enzyme (ACE) act in concert to degrade Bk in the pulmonary circulation. To test this hypothesis, an inhibitor of aminopeptidase P was designed and synthesized. N-[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-prolyl-L-prolyl-L - alaninamide (apstatin) inhibited purified rat lung membrane-bound aminopeptidase P with a Ki value of 2.6 microM (linear mixed-type inhibition, alpha = 5.1, beta = 0). Apstatin did not inhibit ACE or other known Bk-degrading enzymes. Apstatin and an ACE inhibitor, ramiprilat, were tested for their ability to inhibit Bk degradation in the isolated perfused rat lung. [2,3-Proly-3,4-3H(N)]-bradykinin ([3H]-Bk) was perfused through the isolated lung in the presence or absence of inhibitors. The perfusate was then subjected to HPLC to identify and quantitate radiolabeled fragments. In the absence of inhibitors, no intact [3H]-Bk was found in the perfusate. In the presence of ramiprilat (0.5 microM), only 22% +/- 6% of the radioactivity in the perfusate was intact [3H]-Bk, and the remaining radioactivity indicated cleavage of the Arg1-Pro2 bond. When apstatin (40 microM) was perfused along with ramiprilat, degradation of [3H]-Bk was almost completely blocked (92% +/- 4% intact [3H]-Bk in the perfusate). The results indicate that the Bk-degrading activity in the rat pulmonary vascular bed can be fully accounted for by aminopeptidase P (30%) and ACE (70%).

Amino Acid Sequence↗

PubMed1996

[Subcellular distribution of leucine aminopeptidase during the development and aging of rat brains].

INTRODUCTION: The aminopeptidases are considered to be enzymes which can regulate the activity of various neuropeptides. However, their precise function has not yet been fully determined. OBJECTIVE: With a view to studying the function of these enzymes at the cerebral level, the subcellular distribution of soluble leucine aminopeptidase from left and right cerebral hemisphere, in rats of different pre-natal and post-natal ages was determined as described in this paper. MATERIALS AND METHODS: The activity of soluble leucine aminopeptidase was determined fluorimetrically in the sub-cellular fractions obtained from the left and right cerebral hemispheres of rats of different pre-natal and post-natal ages: fetuses (of 19-21 days gestation), 1 week, 1, 5 and 24 months of age. RESULTS: No differences were observed between the cerebral hemispheres at any of the ages studied. However, significant differences were seen in the evolutionary behaviour of the two hemispheres. At all ages, except 24 months, there were significant differences between the fractions. In the fetuses, most activity was seen at the level of the synaptosomal raw fraction and least in the microsomal. At 1-week-old, the highest levels were seen at a microsomal level. In rats of 1 and 5 months of age, the highest levels were detected at synaptosomal level. With regard to the evolution with age, in the homogenate, nuclear fraction, cytosol and mitochondrial fraction a similar profile was observed, with a decrease after birth and an increase at one month, being stable at the other ages. At a synaptosomal level the profile is similar, although the differences are much more marked, with a drastic decrease in activity at two years of age. By contrast, the microsomal fraction showed a very different evolutionary profile with an increase after birth, becoming stable at later ages. CONCLUSIONS: The activity of soluble leucine aminopeptidase shows a heterogeneous subcellular distribution together with significant differences during cerebral development and ageing. The behaviour of this enzyme may reflect the functional state of its neuropeptide substrates.

Age Factors↗

PubMed1996

3-Amino-2-hydroxy-propionaldehyde and 3-amino-1-hydroxy-propan-2-one derivatives: new classes of aminopeptidase inhibitors.

3-Amino-2-hydroxy-propionaldehydes [H2NCH(R)CHOHCHO with R = H, i-Bu, CH2Ph] were designed as metallo-aminopeptidase inhibitors based on the metal active site chelation concept. These compounds were found to be micromolar inhibitors of aminopeptidase-M (AP-M, EC 3.4.11.2) with potencies similar to bestatin (Ki = 3.5 microM). Notably, compound 5a (R = H) is a selective inhibitor of AP-M (Ki = 7 microM) with respect to cytosolic leucine aminopeptidase (LAPc, EC 3.4.11.1) (Ki = 385 microM). However, due to their easy oligomerization, these compounds are low practical value. In contrast, the corresponding isomeric 3-amino-1-hydroxy-propan-2-one derivatives [H2NCH(R)COCH2OH with R = H, i-Bu, CH2Ph, i-Pr, CH2Biph] are well defined structures. These hydroxymethylketones also exhibit micromolar affinities on AP-M. Compound 6c (R = CH2Ph) was the most potent (Ki = 1 microM). Selectivity studies of 6a (R = H) and 6b (R=i-Bu) show a preference for AP-M. Compound 6a is moderately active on AP-M (Ki = 25 microM) and inactive on LAPc. This new class of inhibitors is proposed to bind as bidentates, analogous to hydroxamates.

Aldehydes↗

PubMed2003

Ultrastructural localization of aminopeptidase A/angiotensinase and placental leucine aminopeptidase/oxytocinase in chorionic villi of human placenta.

AIMS: Membrane-bound aminopeptidases in human placenta are thought to be involved in maintaining homeostasis during pregnancy by metabolizing bioactive peptides such as oxytocin and angiotensin at the interface between the fetus and mother. Because determining the precise localization of these enzymes is required to support this notion, we investigated the ultrastructural localization of two principal enzymes, aminopeptidase A (APA; EC 3.4.11.7)/angiotensinase and placental leucine aminopeptidase (P-LAP; EC 3.4.11.3)/oxytocinase in human first trimester and full-term placenta. METHODS: Immunohistochemical analysis using anti-P-LAP and anti-APA antibodies was performed on ultrathin frozen sections of fixed human placental villi. RESULTS: Transmission immunoelectron microscopy revealed that both enzymes were expressed on the surface of apical microvilli of syncytiotrophoblast cells and, to a lesser extent, on the basal infoldings. The location of the two enzymes did not vary between the first trimester and full-term placenta sections, while the staining intensities were slightly enhanced in full-term villi. CONCLUSIONS: Our observation that P-LAP and APA are present on the microvilli, which is a site of interaction between the mother and fetus, suggests possible involvement of these enzymes in cleaving peptide hormones from the fetus and mother in order to regulate bioactivity.

Adult↗

PubMed1999

A bicarbonate ion as a general base in the mechanism of peptide hydrolysis by dizinc leucine aminopeptidase.

The active sites of aminopeptidase A (PepA) from Escherichia coli and leucine aminopeptidase from bovine lens are isostructural, as shown by x-ray structures at 2.5 A and 1.6 A resolution, respectively. In both structures, a bicarbonate anion is bound to an arginine side chain (Arg-356 in PepA and Arg-336 in leucine aminopeptidase) very near two catalytic zinc ions. It is shown that PepA is activated about 10-fold by bicarbonate when L-leucine p-nitroanilide is used as a substrate. No activation by bicarbonate ions is found for mutants R356A, R356K, R356M, and R356E of PepA. In the suggested mechanism, the bicarbonate anion is proposed to facilitate proton transfer from a zinc-bridging water nucleophile to the peptide leaving group. Thus, the function of the bicarbonate ion as a general base is similar to the catalytic role of carboxylate side chains in the presumed mechanisms of other dizinc or monozinc peptidases. A mutational analysis shows that Arg-356 influences activity by binding the bicarbonate ion but is not essential for activity. Mutation of the catalytic Lys-282 reduces k(cat)/K(m) about 10,000-fold.

Aminopeptidases↗

PubMed1992

Increase of methionine aminopeptidase activity in hyperplastic Leydig cells of rat cryptorchid testis: a histochemical study.

Histochemical study on the changes of the aminopeptidase activities in rat testes after surgically-induced cryptorchidism was conducted comparing them with the histochemical changes in regenerated hepatic cells of the partially hepatectomized rat liver. Methionine-aminopeptidase in Leydig cells gradually increased after cryptorchid was induced, whereas the enzyme activity in regenerated hepatic cells decreased. These histochemical observations were coincident with the data obtained by enzyme assay. The present study has indicated that in the rat cryptorchid testis the increase of methionine-aminopeptidase activity was caused by hyperplastic Leydig cells.

Aminopeptidases↗

PubMed2005

CD13 (aminopeptidase N) can associate with tumor-associated antigen L6 and enhance the motility of human lung cancer cells.

Cancer metastasis is a multiple-step process that involves the regulated interaction of diverse cellular proteins. We recently reported that the expression of tumor-associated antigen L6 (TAL6) promoted the invasiveness of lung cancer cells and was inversely correlated with disease-free survival of squamous lung carcinoma patients. We now report that CD13 (aminopeptidase N) can associate with TAL6 and can enhance cancer cell migration. CD13 was shown by coimmunoprecipitation to associate in vitro with TAL6 on several cancer cell lines and to associate in vivo by antibody-mediated copatching immunofluorescence. CD13 was selectively expressed on highly invasive CL1-5 lung cancer cells as compared to poorly invasive CL1-0 lung cancer cells. The role of CD13 aminopeptidase activity in regulating cell motility was investigated with chemical inhibitors, specific antibodies and a catalytically inactive CD13 protein. Inhibition of CD13 aminopeptidase activity by nontoxic concentrations of leuhistin modestly decreased the migration of CL1-5 cells. In contrast, binding of CD13 by specific antibodies significantly reduced both the migration and the invasion of CL1-5 cells. Poorly invasive CL1-0 cells that stably expressed CD13 displayed significantly (p < or = 0.0005) enhanced cell migration (300% of control). Expression of an enzymatically inactive CD13 mutant on CL1-0 cells also significantly (p < or = 0.0005) enhanced cell migration (200% of control). Our results show that TAL6 and CD13 can form a complex on lung cancer cells, that these molecules can modulate cell migration and invasion and that the influence of CD13 on cell motility did not strictly depend on its aminopeptidase activity.

Adenocarcinoma↗

PubMed1995

Regulatory effect of aminopeptidase inhibitor (bestatin) on the cervix during induction of ripening by interleukin-8.

Bestatin is an immunomodulatory peptide that stimulates the humoral and cell-mediated immune system. It also has an inhibitory effect on multiple aminopeptidases. Recently we found that aminopeptidase N inactivates interleukin-8 in vitro. Bestatin successfully suppresses the effect of aminopeptidase N on interleukin-8. During cervical maturation many biochemical changes occur including decrease in collagen concentration and increase in collagenase and elastase activities. Interleukin-8, which has a potent neutrophil chemotactic effect, was found to induce cervical ripening in rabbits. The combination of interleukin-8 with bestatin also induced cervical ripening by providing approximately regular levels of neutrophil numbers, collagenase, and elastase activities. We therefore suggest that this regulatory mechanism also takes place in vivo through the inhibitory effect of bestatin on aminopeptidase N.

Animals↗

PubMed1995

Aminopeptidase N: a constitutive cell-surface protein on human dermal fibroblasts.

Monoclonal antibodies were developed against human dermal fibroblast cell-surface membranes. Our goal was to find evidence of differential expression of antigens on fibroblast clones derived by limiting dilution. Antibodies were then used to isolate and identify membrane proteins. By sequence analysis of membrane immunoprecipitates, one monoclonal antibody, BR2, was subsequently shown to recognize the enzyme aminopeptidase N (hAPN; EC 3.4.11.2), originally described as a marker for certain hematopoetic cells. We have used biochemical techniques, electron microscopy, flow cytometry, and ELISA to characterize aminopeptidase N expression by human dermal fibroblasts. The presence of abundant aminopeptidase N confers enzymatic properties to human dermal fibroblasts which heretofore have been largely unexplored and suggest that the cellular distribution of aminopeptidase N is wider than originally appreciated.

Antibodies, Monoclonal↗

PubMed2001

Aminopeptidase-N from the Helicoverpa armigera (Hubner) brush border membrane vesicles as a receptor of Bacillus thuringiensis crylac delta-endotoxin.

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type delta-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2'-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of delta-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60-77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.

Amino Acid Sequence↗

PubMedDate not supplied

Alanine aminopeptidase of guinea-pig brain: a broad specificity cytoplasmic enzyme capable of hydrolysing short and intermediate length peptides.

Alanine aminopeptidase is reported to be a broad specificity aminopeptidase acting on peptides of different lengths. In this study we wish to define the properties of the activity from guinea-pig brain and compare these properties with previous findings. Alanine amino-peptidase was purified from cytoplasm of guinea-pig brain by a four-step procedure involving chromatography on DE-52, hydroxylapatite, Sephacryl S-200 and DEAE-Sephacryl. Relative molecular mass was determined by chromatography on Sephacryl S-200 column and subunit size determined by SDS-PAGE under denaturing conditions. Cations which reactivate the enzyme were determined with EDTA treated enzyme. Substrate specificity was determined by TLC and kinetic parameters were derived from Lineweaver-Burk plots. A 216-fold purification was achieved by the above procedures. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 104,000. Its activity was inhibited by chelating agents, sulphydryl reactive agents, puromycin, bestatin and amastatin but stimulated over 6-fold by dithiothreitol. Some dipeptides and all tripeptides and longer peptides containing up to 16 amino acids tested were hydrolysed provided neither Glp or Pro occurred at the N-terminus or that Pro did not occur in the penultimate position from the N-terminus. The enzyme preferred bulky non-polar residues at the N-terminal and penultimate positions and was found to hydrolyse three dipeptidyl methyl coumarin amides used in detecting dipeptidyl aminopeptidases. Alanine aminopeptidase is thus a broad specificity amino-peptidase acting on short and intermediate length peptides whose affinity for substrates increases with increasing peptide length. Its properties are well suited to a role in peptide turnover in brain cytoplasm.

Amino Acid Sequence↗

PubMed1995

A study of a synaptosomal thyrotropin releasing hormone-inactivating pyroglutamate aminopeptidase from bovine brain.

Pyroglutamate aminopeptidase type II is a highly specific membrane-bound neuropeptidase that has the ability to remove N-terminal pyroglutamate (Glp) from Thyrotropin Releasing Hormone (Glp-His-Pro-NH2) or very closely related tripeptides or tripeptide amides. In this paper we report on the purification and characterisation of a pyroglutamate aminopeptidase activity from the synaptosomal membranes of bovine brain. The Triton X-100 solubilised enzyme was purified nearly 600-fold by a combination of conventional column chromatography steps with a recovery/yield of 17.0%. Phase-partitioning experiments with Triton X-114 showed the activity to be an integral membrane protein. This detergent-solubilised pyroglutamate aminopeptidase activity was found to have a relative molecular mass of 240 kDa on a calibrated S-200 column. HPLC analysis on a C18 reverse-phase column showed that the purified activity displayed a very narrow substrate specificity cleaving only Thyrotropin Releasing Hormone (TRH) or the very closely related acid-TRH, LHRH (1-3) and the TRH-analogue (methyl-His)-TRH and had a Km of 100 microM for the fluorimetric substrate Glp-His-Pro-methyl-coumarin. The enzyme was inactivated by the metalchelator 1,10-ortho-phenanthroline but showed less sensitivity to EDTA. It also showed some inhibition by thiol protease inhibitors such as iodoacetate and n-ethyl-maleimide. In summary, we have purified a pyroglutamate aminopeptidase from the synaptosomal membrane of bovine brain. This enzyme displays characteristics consistent with it being classified as a PAP type II neuropeptidase with only minor differences from other proteases in this group.

Amino Acid Sequence↗

PubMed1998

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.

The molecular components ensuring the strict exopeptidase action of aminopeptidase N (APN) and related zinc aminopeptidases of the M1 family have not yet been clearly established. The specific recognition of the N-terminal amino acid of the substrates by the enzymes has been proposed to involve either the complexation of the free amino group by the catalytic zinc ion or an interaction with an anionic binding site, which could be constituted by an aspartate or glutamate residue. To investigate the existence of such an ionic binding site, site-directed mutagenesis experiments have been performed on acidic residues of pig APN. Given that aminopeptidases of the M1 family are likely to have a common mechanism of action, only strictly conserved residues were mutated. As compared to the wild-type enzyme, the mutation D220E led only to slight modifications in the kinetic parameters of the enzyme and in the Ki values of various inhibitors, indicating that this residue is not critically involved in the hydrolytic mechanism. In contrast, the mutations E350Q and E350D induced a large decrease in enzyme activity, essentially due to modifications in kcat, whereas the E350A mutation led to an almost completely inactive enzyme. Moreover, among the inhibitors tested, only those acting as transition state analogs showed significant increases in their Ki values. These data are in favor of E350 belonging to the "anionic binding site" in APN. A mechanism of action, derived from that of thermolysin, is proposed for these aminopeptidases, which explains the importance of E350 in transition state formation, rather than in the Michaelis complex.

Amino Acid Sequence↗

PubMed1997

Novel bioadhesive chitosan-EDTA conjugate protects leucine enkephalin from degradation by aminopeptidase N.

PURPOSE: To develop a novel bioadhesive polymer that protects peptide drugs from luminal degradation by aminopeptidase N and to evaluate the system in vitro on porcine mucosa. METHODS: EDTA was covalently bound to chitosan in order to combine the bioadhesive properties of the polymer with the well known capacity of EDTA to complexed metal ions which are essential for the enzymatic activity of proteases. The inhibitory effect of this polymer conjugate was evaluated by using leucine enkephalin (Leu enkephalin) as a model drug. The degree of Leu enkephalin degradation caused by aminopeptidase N (EC 3.4.11.2), as well as porcine mucosa, in the presence of the polymer conjugate, was quantified by HPLC analysis. RESULTS: The chitosan-EDTA conjugate is capable of binding 2.01 +/- 0.12 mmole of zinc per gram of polymer at pH 6.5 (n = 3; +/-S.D.). As zinc is an essential co-factor for aminopeptidase N, enzyme activity (48 mU/ml) could be completely inhibited under the use of 1.0% chitosan-EDTA conjugate. The inhibitory effect of 1.0% chitosan-EDTA conjugate on the degradation of Leu enkephalin on porcine mucosa within 3 h at 37 degrees C was even 2.9-fold higher than that of a recently developed zinc complexing bacitracin-poly(acrylic acid) conjugate of the same concentration. The novel polymer conjugate is more bioadhesive than unmodified chitosan and is easily hydratable in water and basic aqueous solutions exhibiting quick swelling properties. CONCLUSIONS: The bioadhesive polymer conjugate described here seems to be a useful tool in overcoming enzymatic degradation by aminopeptidase N.

Adhesives↗