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PubMed1996

Bacterial aminopeptidases: properties and functions.

Aminopeptidases are exopeptidases that selectively release N-terminal amino acid residues from polypeptides and proteins. Bacteria display several aminopeptidasic activities which may be localised in the cytoplasm, on membranes, associated with the cell envelope or secreted into the extracellular media. Studies on the bacterial aminopeptide system have been carried out over the past three decades and are significant in fundamental and biotechnological domains. At present, about one hundred bacterial aminopeptidases have been purified and biochemically studied. About forty genes encoding aminopeptidases have also been cloned and characterised. Recently, the three-dimensional structure of two aminopeptidases, the methionine aminopeptidase from Escherichia coli and the leucine aminopeptidase from Aeromonas proteolytica, have been elucidated by crystallographic studies. Most of the quoted studies demonstrate that bacterial aminopeptidases generally show Michaelis-Menten kinetics and can be placed into either of two categories based on their substrate specificity: broad or narrow. These enzymes can also be classified by another criterium based on their catalytic mechanism: metallo-, cysteine- and serine-aminopeptidases, the former type being predominant in bacteria. Aminopeptidases play a role in several important physiological processes. It is noteworthy that some of them take part in the catabolism of exogenously supplied peptides and are necessary for the final steps of protein turnover. In addition, they are involved in some specific functions, such as the cleavage of N-terminal methionine from newly synthesised peptide chains (methionine aminopeptidases), the stabilisation of multicopy ColE1 based plasmids (aminopeptidase A) and the pyroglutamyl aminopeptidase (Pcp) present in many bacteria and responsible for the cleavage of the N-terminal pyroglutamate.

Amino Acid Sequence↗

PubMed1965

BITTER PEPTIDE ISOLATED FROM MILK CULTURES OF STREPTOCOCCUS CREMORIS.

Certain cultures of Streptococcus cremoris produced a bitter taste that occurred in the whey portion of milk cultures. Whey from a culture which produced bitterness was fractionated on Sephadex. The fraction in which the bitter taste was concentrated was chromatographed successively on paper with butanol-acetic acid-water (5:1:4), and then butanol-2-butanone-water (2:2:1). In each instance, the bitter component was in the most rapidly moving band that gave a positive ninhydrin test. The bitterness was observed to be caused by a peptide containing the following numbers of each amino acid: arginine, 1; glutamic acid, 2; glycine, 2; isoleucine, 2; leucine, 2; phenylalanine, 1; proline, 5; and valine, 4. N-terminal amino acids could be detected by coupling with 2,4-dinitrofluorobenzene or phenylisothiocyanate, or by hydrolysis with leucine aminopeptidase. When treated with carboxypeptidase, only leucine and valine appeared at the C-terminal end, and these were detected simultaneously.

Alanine↗

PubMed2005

alpha-Aminoalkylphosphonates as a tool in experimental optimisation of P1 side chain shape of potential inhibitors in S1 pocket of leucine- and neutral aminopeptidases.

The synthesis and biological activity studies of the series of structurally different alpha-aminoalkylphosphonates were performed in order to optimise the shape of the side chain of the potential inhibitors in S1 pocket of leucine aminopeptidase [E.C.3.4.11.1]. Analysis of a series of compounds with aromatic, aliphatic and alicyclic P1 side chains enabled to find out the structural features, optimal for that fragment of inhibitors of LAP. The most active among all investigated compounds were the phosphonic analogues of homo-tyrosine (K(i)=120 nM) and homo-phenylalanine (K(i)=140 nM), which even as racemic mixtures were better inhibitors in comparison with the best till now-phosphonic analogue of l-leucine (230 nM). Additional comparison of the inhibitory activity obtained for aminopeptidase N (APN, E.C.3.4.11.2) give insight into structural preferences of both enzymes.

Aminopeptidases↗

PubMed2005

Nephrotoxicity of organic solvents: biomarkers for early detection.

OBJECTIVES: Evidence for a relationship between chronic kidney diseases or progression of already existing diseases (glomerulonephritides) and occupational solvent exposure has been found in case reports, in case-control studies and also in cross-sectional studies. An analysis of the available literature was performed with respect to markers measured in cross-sectional studies that might be useful for an early detection of solvent-induced effects on the kidney. METHODS: The relevant cross-sectional studies were evaluated and the following markers were analyzed with respect to their suitability as biomarker for renal damage: total protein, albumin, transferrin, IgG, beta(2)-microglobulin, retinol-binding protein, N-acetyl-beta-D: -glucosaminidase, alanine aminopeptidase, beta-galactosidase, beta-glucuronidase, leucin aminopeptidase, alkaline phosphatase, lysozyme, Tamm-Horsfall protein and laminin fragments in urine as well as E-selectin, laminin and anti-laminin antibodies and anti-glomerular basement membrane antibodies in serum. RESULTS: An increased albumin excretion was observed more frequently in groups of workers exposed to various solvents (like toluene, styrene, aliphatic/aromatic hydrocarbon mixtures, tetrachloroethene, mixtures of chlorinated hydrocarbons) than in controls. No clear pattern emerged for the other markers. CONCLUSIONS: The determination of albumin excretion in the urine appears to be a useful parameter for monitoring solvent-exposed workers.

Biomarkers↗

PubMed1996

Breakdown of peptides from a casein hydrolysate by rumen bacteria. Simultaneous study of enzyme activities and physicochemical parameters.

The breakdown of a pancreatic hydrolysate of casein (tryptone) by an inoculum of ruminal mixed bacteria was studied in vitro. Peptides were degradated at 33% after 5 h. The dipeptidyl aminopeptidase type 1 (DAP-1), exoaminopeptidase and leucine aminopeptidase (LAP) like activities were measured using, respectively, Gly-Arg-MNA, Ala-pNa and Leu-pNA as substrates. While the total proteolytic activity remained stable throughout peptide breakdown, the DAP-1 already present at the beginning of fermentation increased until two times its early activity. This peptidase activity is a major index for the presence of Prevotella ruminicola. A peak of exoaminopeptidase and LAP activities were observed at 3 h incubation. This latter activity, which was associated with the production of lactate after 3 h, suggested that Streptococcus bovis was present in the environment. With the use of high-performance size exclusion chromatography (HPSEC), the size of the peptides during their breakdown could be measured, showing that high molecular weight peptides (4 to 2 kDa) were apparently more rapidly broken down to the profit of small-sized peptides (< 0.5 kDa), which increased. These degradation abilities were closely linked to DAP-1 activity. Separation of tryptone peptides by reverse-phase high-performance liquid chromatography (HPLC) showed that more hydrophilic peptides disappeared than hydrophobic peptides. Moreover, a few peaks eluting for one in the hydrophilic area and for the others in the hydrophobic area resisted the bacterial breakdown. In contrast, amino acid profiles indicated that hydrophobic amino acids were not taken up significantly faster than the hydrophobic amino acids by mixed ruminal bacterial.

Amino Acids↗

PubMed1975

Studies of the distribution of proteins bound to CNBr-activated Sepharose 6B at the electron-microscopic level.

Proteins were covalently attached to Sepharose by the CNBr method. Their distribution across the carrier beads was studied at the electron microscopic level. The approach has been to ferritinstain and to section the gel beads. Ferritin was either coupled directly to the polysaccharide backbone of the carrier or conjugated with pure rabbit anti-aminopeptidase in order to visualize covalently bound leucine aminopeptidase by the immunferritin technique. The results corroborate earlier fluorescence microscopic findings of a uniform protein distribution, provided that a number of conditions are fulfilled.

Binding Sites↗

PubMed1996

Enzyme-triggered formation of electrochemiluminescent ruthenium complexes.

A sensitive enzyme assay is described using substrates derivatized with metal-binding ligands. Enzymes catalyzed changes in the abilities of substrates to bind to the nonelectrochemiluminescent complex ruthenium (II) bis(bipyridyl), Ru(bpy)2(2+), to form electrochemiluminescent mixed-ligand complexes. A highly electrochemiluminescent complex was formed between Ru(bpy)2(2+) and picolinic acid (1) but not picolinic acid ethyl ester (4), permitting the detection of 4 hydrolysis by pig liver esterase (PLE). Electrochemiluminescence (ECL) differences between Ru(bpy)2(2+) mixtures of 1 and 4 were detected to a lower concentration limit of 65pM. Under the conditions used in actual enzyme assays, it was possible to detect 4.4 pM PLE and the hydrolysis of 1.3 microM 4. In a second assay, leucine aminopeptidase (LAP) hydrolyzed 8-(L)-leucylaminoquinoline (9) to leucine and aminoquinoline (10). A mixed-ligand complex formed between (9) and Ru(bpy)2 (2+) was substantially more electrochemiluminescent than a complex of Ru(bpy)2(2+) and 10. ECL differences between Ru(bpy)2(2+) mixtures of 9 and 10 were detectable to 65 nM. Under actual enzyme assay conditions, 375 pM LAP could be detected as well as the hydrolysis of 1.3 microM 9.

2,2'-Dipyridyl↗

PubMed2005

Characterisation of Aspergillus niger prolyl aminopeptidase.

We have cloned a gene (papA) that encodes a prolyl aminopeptidase from Aspergillus niger. Homologous genes are present in the genomes of the Eurotiales A. nidulans, A. fumigatus and Talaromyces emersonii, but the gene is not present in the genome of the yeast Saccharomyces cerevisiae. Cell extracts of strains overexpressing the gene under the control of its own promoter showed a fourfold to sixfold increase in prolyl aminopeptidase activity, but no change in phenylalanine or leucine aminopeptidase activity. The overexpressed enzyme was subsequently purified and characterised. The enzyme specifically removes N-terminal proline and hydroxyproline residues from peptides. It is the first enzyme of its kind from a eukaryotic organism that has been characterised.

Amino Acid Sequence↗

PubMed1995

Cloning and sequence analysis of the aminopeptidase My gene from Mycoplasma salivarium.

The complete nucleotide sequence of a major component of aminopeptidase My purified from Mycoplasma salivarium was determined. The protein gene encoded a protein consisting of 520 amino acids with a molecular mass of 58079 Da. The protein contained two tryptophan residues, one of which was encoded by UGA. A computer-aided homology search suggested that aminopeptidase My had properties similar to those of leucine aminopeptidase (EC 3.4.11.1).

Amino Acid Sequence↗

PubMed2001

26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide.

Protein degradation by proteasomes is the source of most antigenic peptides presented on MHC class I molecules. To determine whether proteasomes generate these peptides directly or longer precursors, we developed new methods to measure the efficiency with which 26S and 20S particles, during degradation of a protein, generate the presented epitope or potential precursors. Breakdown of ovalbumin by the 26S and 20S proteasomes yielded the immunodominant peptide SIINFEKL, but produced primarily variants containing 1-7 additional N-terminal residues. Only 6-8% of the times that ovalbumin molecules were digested was a SIINFEKL or an N-extended version produced. Surprisingly, immunoproteasomes which contain the interferon-gamma-induced beta-subunits and are more efficient in antigen presentation, produced no more SIINFEKL than proteasomes. However, the immunoproteasomes released 2-4 times more of certain N-extended versions. These observations show that the changes in cleavage specificity of immunoproteasomes influence not only the C-terminus, but also the N-terminus of potential antigenic peptides, and suggest that most MHC-presented peptides result from N-terminal trimming of larger proteasome products by aminopeptidases (e.g. the interferon-gamma-induced enzyme leucine aminopeptidase).

Animals↗

PubMed2004

Regulatory role of membrane-bound peptidases in the progression of gynecologic malignancies.

Membrane-bound peptidases play a key role in the control of growth, differentiation, and signal transduction of many cellular systems by degrading bioactive peptides. Thus, abnormal changes in their expression pattern and catalytic function result in altered peptide activation, which contributes to neoplastic transformation or progression. In this review, we describe our recent findings along with work from other groups on the expression and biological functions of membrane-bound peptidases in cancer, focusing on the regulatory roles of three peptidases, aminopeptidase A (APA), neutral endopeptidase (NEP) and placental leucine aminopeptidase (P-LAP), in the progression of gynecologic malignancies. APA, NEP and P-LAP are differentially expressed and localized in various gynecologic malignancies including cervical cancer, endometrial cancer, ovarian cancer and choriocarcinoma in a tumor-type specific pattern. The expression levels are up- or down-regulated depending on histological grade or disease progression. These peptidases play regulatory roles in tumor cell proliferation, invasion or angiogenesis via degradation/inactivation of target peptides such as angiotensin II, endothelin-1 and oxytocin, which act on cancer cells as stimulatory or inhibitory factors. Thus, membrane-bound peptidases may become not only a new diagnostic/prognostic marker, but also a novel molecular target for the treatment of gynecologic malignancies.

Disease Progression↗

PubMed1976

[Protein hydrolysis by immobilized enzymes].

The enzymic hydrolysis of some proteins (insulin-B-chain-S-sulfonate, S-aminoethylated lysozyme, bovine serum albumin) by immobilized peptidolytic enzymes is reported. Sepharose-bound pronase, trypsin and a protease from Thermoactinomyces sp. (MP), the latter both cross linked by glutaric dialdehyde and an exopeptidase mixture containing Sepharose-bound leucine aminopeptidase, carboxypeptidase A and a crude preparation of prolidase were used. After enzymic hydrolysis nearly all amino acids, except proline, were recovered in a 100% yield compared to the value of an acid reference hydrolysate. Tryptophan and methionine, which are partially destroyed by acid hydrolysis in the presence of oxygen could be recovered completely.

Aminopeptidases↗

PubMed1970

Naphthylamidase activity of Leptospira.

Extracts of 18 serotypes of the genus Leptospira were found to possess naphthylamidase activity, and differences in the pathogenic and saprophytic strains were noted. The former exhibited a preference for the leucyl naphthylamide substrate, whereas the latter demonstrated greater hydrolysis of alanyl naphthylamide. With the leucyl naphthylamide as substrate, pathogenic strains showed 10 to 20 times higher naphthylamidase activity than saprophytic strains. Optimal temperature and pH for enzymatic hydrolysis also differed between pathogenic and saprophytic strains. Maximal enzymatic activities for pathogenic and saprophytic naphthylamidases were 41 and 37 C, respectively, at pH 8.0 to 8.5. The pH and temperature optima suggested that the leptospiral enzyme activity was not leucine aminopeptidase.

Alanine↗

PubMed2002

Characterization of a cytosolic aminopeptidase from Encephalitozoon intestinalis.

Aminopeptidase activity was detected in Encephalitozoon intestinalis using a fluorometric assay. The aminopeptidase was capable of hydrolysing different amino acids bound to 7-amino-4-trifluoromethyl coumarin, with maximal activity against the amino acid, leucine. Aminopeptidase activity was localized in E. intestinalis spores and in intracellular stages. Enzymatic activity was inhibited by the traditional aminopeptidase inhibitors, bestatin and its analogue, nitrobestatin. Inhibition with the chelating agents, EDTA and 1,10-phenanthroline, suggested that the enzyme activity belongs to the metalloaminopeptidase class. Subcellular fractionation demonstrated that maximal enzyme activity was localized in the cytosolic fraction. Direct fluorogenic substrate analysis by native polyacrylamide gel electrophoresis estimated a molecular weight of 70.8 kDa. Direct fluorogenic analysis by polyacrylamide ampholyte gel electrophoresis indicated an isoelectric point of 4.8. The enzyme was both heat (> 37 degrees C) and cold (< 0 degrees C) labile with an optimal activity at pH 7.2. This is the first report characterizing a cytosolic aminopeptidase in microsporidia.

Animals↗

PubMed1975

[Effect of Solanum melongena on experimental atheromatosis. VI. Enzyme histochemical, physiopathological and chemical studies on cholesterol-induced atheromatosis in rabbits. Conclusions (author's transl)].

QUESTION: The mode of action of Solanum Melongena (Sol. Mel.) in its inhibitory effect on the development of atheromatous plaques in cholesterol-induced atheromatosis in rabbits is still unexplained. In this paper the author reports his enzyme histochemical, physiopathological and chemical observations drawing conclusions for future experiments. MATERIALS AND METHODS: Animals and experimental conditions were the same as described in the recent studies (see literature no. 2, 15, 16, 17, 18). 1. Enzyme histochemistry: The aortas were removed immediately after death, placed in Holt's solution, freed from the advential fatty tissue, cut into 5-6 mm high cylinders and either processed instantly or preserved in the same fluid at +2 degrees C. From the individual portions cryostat sections (10 mum thick) were cut after the method of Pearse. They were mounted to cover glasses (22 X22) and placed in substrate-filled specially produced cuvettes. The capacity of such a cuvette was 10 ml with room for 4 cover glasses each 4 mm apart. For protection against vaporization small caps of plastic foil were used. The method given by GLOCKNER and SCHMIDT (7) who recently reported their studies in the rat aorta, yielded satisfactory results in small arteries. In thereabbit aorta, however, the results varied markedly, which may be due to greater thickness of the vascular wall. Consequently, despite of all its advantages, this technique could not be employed in this study. The following enzymes were demonstrated: hydrolases: phosphomonoesterase I--3.1.3.1. with naphthol AS-sulphate after BURSTONE in the modification of LOJDA phosphomonoesterase II--3.1.3.2. after BURSTONE as modified by LOJDA with naphthol AS-phosphate and hexazonium pararosanilin leucine aminopeptidase--3.4.1.1. technique according to NACHLAS et al. with leucine beta-naphthylamide lipase--3.1.1.3. after GOMORI (Tween 80) adenosine-5-phosphatase (5'nucleotidase)--3.1.3.5. after GOMORI succinic dehydrogenase--1.3.99.1.--method according to NACHLAS et al. NAD (NADH and NADP (NADPH) diaphorase--1.4.1.2.--1.4.1.4., technique of SCARPELLI, HESS and PEARSE as modified by LOJDA (see article) Substratefree or heatinactivated controls were prepared for each histochemical demonstration. Several additional series with phenazine methosulphate (PMS) as an intermediate electron acceptor or such without PMS as well as preparations with KCN or without KCN were examined for the demonstration of anaerobic dehydrogenases. However, to avoid the nothing dehydrogenase effect only PMS-processed specimens were evaluated. For histological information several additional sections of the tissue samples were stained with haematoxylin-eosin or with elastica-van Gieson and Weigert's iron haematoxylin in combination. The enzyme histochemical preparations were embedded in Kaiser's glycerine gelatin, the histological sections were embedded in Caedox. The test animals were grouped like in the recent experiments (see literature no. 2, 15, 16, 17, 18). 2...

Animals↗

PubMed1997

Peripherally active analgesia of aminopeptidase-resistant sugar-coupled leucine enkephalin.

Aminopeptidase-degradable leucine enkephalin (LE) was coupled with disaccharide (cellobiose or gentiobiose). Acetic acid writhing assay (for peripheral effect) showed that cellobiose-coupled LE (CcpLE) as well as LE had analgesic activity by subcutaneous (s.c.) administration to mice pretreated (s.c. administration) with peptidase inhibitors, whereas any analgesic effect was not observed after s.c. administration of CcpLE or LE without peptidase inhibitors. However, gentiobiose-coupled LE (EcpLE) did not have analgesic activity even with pretreatment of peptidase inhibitors. On the other hand, hot-plate assay (for central effect) did not indicate obvious analgesic effect after s.c. administration of CcpLE or EcpLE with pretreatment of peptidase inhibitors. Significant difference of stability against peptidase was not observed between CcpLE and EcpLE. Therefore, it was concluded that CcpLE is a peripherally active analgesic.

Aminopeptidases↗

PubMed2002

Mice deficient in the insulin-regulated membrane aminopeptidase show substantial decreases in glucose transporter GLUT4 levels but maintain normal glucose homeostasis.

The insulin-regulated aminopeptidase (IRAP) is a zinc-dependent membrane aminopeptidase. It is the homologue of the human placental leucine aminopeptidase. In fat and muscle cells, IRAP colocalizes with the insulin-responsive glucose transporter GLUT4 in intracellular vesicles and redistributes to the cell surface in response to insulin, as GLUT4 does. To address the question of the physiological function of IRAP, we generated mice with a targeted disruption of the IRAP gene (IRAP-/-). Herein, we describe the characterization of these mice with regard to glucose homeostasis and regulation of GLUT4. Fed and fasted blood glucose and insulin levels in the IRAP-/- mice were normal. Whereas IRAP-/- mice responded to glucose administration like control mice, they exhibited an impaired response to insulin. Basal and insulin-stimulated glucose uptake in extensor digitorum longus muscle, and adipocytes isolated from IRAP-/- mice were decreased by 30-60% but were normal for soleus muscle from male IRAP-/- mice. Total GLUT4 levels were diminished by 40-85% in the IRAP-/- mice in the different muscles and in adipocytes. The relative distribution of GLUT4 in subcellular fractions of basal and insulin-stimulated IRAP-/- adipocytes was the same as in control cells. We conclude that IRAP-/- mice maintain normal glucose homeostasis despite decreased glucose uptake into muscle and fat cells. The absence of IRAP does not affect the subcellular distribution of GLUT4 in adipocytes. However, it leads to substantial decreases in GLUT4 expression.

Adipocytes↗

PubMed2004

Role of the insulin-regulated aminopeptidase IRAP in insulin action and diabetes.

The insulin-regulated aminopeptidase (IRAP) is a member of the family of zinc-dependent membrane aminopeptidases. It is the homolog of the human placental leucine aminopeptidase (P-LAP). IRAP is expressed in different cell types but has been best characterized in two major insulin target cells, muscle and fat. In these cells IRAP localizes to intracellular membrane compartments under basal conditions. In response to insulin IRAP redistributes to the cell surface. IRAP shares this behavior with the insulin-responsive glucose transporter GLUT4. It is established that insulin's dramatic effect on glucose disposal is mediated through its action on GLUT4. The role IRAP plays in insulin action is unknown. IRAP cleaves several peptide hormones in vitro. In insulin-treated cells, concomitant with the appearance of IRAP at the cell surface, aminopeptidase activity toward extracellular substrates increases. Thus, insulin, by bringing IRAP to the cell surface, could increase the processing of extracellular peptide hormones and thereby change their activities. Investigations are underway to determine the in vivo substrates for IRAP and to measure the effect of insulin on the cleavage of identified substrates. In individuals with type 2 diabetes the insulin-stimulated translocation of IRAP to the cell surface of muscle and fat cells is impaired. This defect may lead to decreased cleavage and consequently increased action of peptide hormones that are substrates for IRAP. Impaired IRAP action may thus play a role in the development of complications in type 2 diabetes. The findings of decreased expression of GLUT4 and increased heart size in mice in which IRAP was deleted support this hypothesis.

Amino Acid Sequence↗