Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “AMINOPEPTIDASES”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,333 records · Page 74Linked to original sources
PubMed2005

Analysis and expression of STE13ca gene encoding a putative X-prolyl dipeptidyl aminopeptidase from Candida albicans.

Candida albicans STE13ca gene was identified by its homology to the Saccharomyces cerevisiae STE13 gene that encodes for the dipeptidyl aminopeptidase A (DAP A) involved in the maturation of alpha-factor mating pheromone. Our study revealed that C. albicans ATCC 10231 depicts dipeptidyl aminopeptidase activity. We also analyzed the expression of the STE13ca gene homologue from this pathogenic yeast. This gene of 2793 pb is homozygotic and encodes for a predicted protein of 930 amino acids with a molecular weight of 107,035 Da. The predicted protein displays significant sequence similarity to S. cerevisiae Ste13p. This C. albicans gene is located in chromosome R. STE13ca gene increases its levels of expression in conditions of nutritional stress (proline as nitrogen source) and during formation of the germinal tube, suggesting a basic biological function for the STE13ca in this yeast.

Amino Acid Sequence↗
PubMed2004

Structure of a human A-type potassium channel interacting protein DPPX, a member of the dipeptidyl aminopeptidase family.

It has recently been reported that dipeptidyl aminopeptidase X (DPPX) interacts with the voltage-gated potassium channel Kv4 and that co-expression of DPPX together with Kv4 pore forming alpha-subunits, and potassium channel interacting proteins (KChIPs), reconstitutes properties of native A-type potassium channels in vitro. Here we report the X-ray crystal structure of the extracellular domain of human DPPX determined at 3.0A resolution. This structure reveals the potential for a surface electrostatic change based on the protonation state of histidine. Subtle changes in extracellular pH might modulate the interaction of DPPX with Kv4.2 and possibly with other proteins. We propose models of DPPX interaction with the voltage-gated potassium channel complex. The dimeric structure of DPPX is highly homologous to the related protein DPP-IV. Comparison of the active sites of DPPX and DPP-IV reveals loss of the catalytic serine residue but the presence of an additional serine near the "active" site. However, the arrangement of residues is inconsistent with that of canonical serine proteases and DPPX is unlikely to function as a protease (dipeptidyl aminopeptidase).

Alternative Splicing↗
PubMed2005

Regulation of aminopeptidase N (EC 3.4.11.2; APN; CD13) by interferon-gamma on the HL-60 cell line.

Membrane-bound peptidases play important roles in the regulation of local concentrations of various signalling peptides such as the growth factors, hormones, chemokines and cytokines. That is accomplished by means of their enzyme activity. Recently, membrane-bound peptidases have also been shown to act as receptors, receiving signals from as yet undefined ligands and transducing them into the cell interior. By using either or both of these mechanisms, peptidases interact with fundamental cellular functions: growth, differentiation, activation and death. This study addressed the effects of a T-cell derived cytokine, interferon-gamma (IFN-gamma) on the activity of aminopeptidase N (APN), an ectoenzyme processing several signal peptides. Cells of a myelo-monocytic cell line HL-60 were used as a model system, and APN was assayed at the levels of mRNA, its membrane marker CD13, and the enzyme activity. Regulation of CD13/APN by IFN-gamma was found at all three levels. The direction of regulation was time-dependent: an initial down-regulation seen 24 and 48 hrs after the onset of treatment with IFN-gamma was replaced by an up-regulation after 72 and/or 96 hrs. Up-regulation of CD13/APN observed after 96 hrs was preceded by an up-regulation of APN mRNA reaching its maximum after 72 hrs. The IFN-gamma-induced regulation of APN was due to membrane aminopeptidase N, since it could be completely abrogated by an APN blocking antibody WM-15. The delayed up-regulation of CD13/APN (observed after 72 and/or 96 hrs), required de novo protein synthesis as it could be abrogated by cycloheximide, an inhibitor of protein synthesis. Possible role of endogenous (IFN-gamma-induced) TGF-beta in mediating CD13/APN up-regulation could be excluded, since no TGF-beta was found in supernatants of IFN-gamma treated HL-60 cells. Thus, our data show regulation of CD13/APN on cells of myelo-monocytic origin by a T-cell derived cytokine, IFN-gamma. A similar mechanism might play a role in inflammation.

Antineoplastic Agents↗
PubMed1999

Nature of the anchors of membrane-bound aminopeptidase, amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells.

Spodoptera frugiperda larvae have a microvillar aminopeptidase and both soluble and membrane-bound forms of amylase and trypsin. Membrane-bound aminopeptidase is solubilized by glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) and detergents, suggesting it has a GPI anchor. Membrane-bound trypsin is not affected by GPI-PLC, although it is solubilized by papain and by different detergents. Membrane-bound amylase is similar to trypsin, although once solubilized in detergent it behaves as a hydrophilic protein. Musca domestica trypsin antiserum cross-reacts with only one polypeptide from S. frugiperda midgut. With this antiserum, trypsin was immunolocalized in the anterior midgut cells at the microvillar surface and on the membranes of secretory vesicles found in the apical cytoplasm and inside the microvilli. The data suggest that in this region trypsin is bound to the secretory vesicle membrane by a hydrophobic anchor. Vesicles migrate through the microvilli and are discharged into the lumen by a pinching-off process. Trypsin is then partly processed to a soluble form and partly, still bound to vesicle membranes, incorporated into the peritrophic membrane. In posterior midgut cells, trypsin immunolabelling is randomly distributed inside the secretory vesicles and at the microvilli surface, suggesting exocytosis. Amylase probably follows a route similar to that described for trypsin in anterior midgut, although membrane-bound forms (peptide anchor) solubilize apparently as a consequence of a pH increase inside the vesicles.

Journal Article↗
PubMed1996

Immunoelectron microscopic single and double labelling of aminopeptidase N (CD 13) and dipeptidyl peptidase IV (CD 26).

Using ultrathin cryosections and immunogold labelling, aminopeptidase N (CD 13) and dipeptidyl peptidase IV (CD 26) were localized on the luminal side of the brush border membrane of proximal tubular cells in human kidney as well as of enterocytes from rat small intestine. Furthermore, both enzymes could be detected on the cell surface of human T lymphocytes and especially aminopeptidase N on human synovial fibroblasts. Gold labelled vesicular structures were also found in the cytoplasm in the apical part of renal proximal tubular cells and synovial fibroblasts. In human kidney the colocalization of the two membrane antigens was possible by using several double labelling methods.

Animals↗
PubMed1999

Molecular cloning and expression in Escherichia coli of the extracellular endoprotease of Aeromonas caviae T-64, a pro-aminopeptidase processing enzyme(1).

PA protease (pro-aminopeptidase processing protease) activates the pro-aminopeptidases from Aeromonas caviae T-64 and Vibrio proteolytica by removal of their pro-regions. Cloning and sequencing of the PA protease gene revealed that PA protease was translated as a preproprotein consisting of four domains: a signal peptide; an N-terminal propeptide; a mature region; and a C-terminal propeptide. The deduced amino acid sequence of the PA protease precursor showed significant homology with several bacterial metalloproteases. Expression of the PA protease gene in Escherichia coli indicated that the N-terminal propeptide of the PA protease precursor is essential to obtain the active form of the protease. The N- and C-terminal propeptides of the expressed pro-PA protease were processed autocatalytically.

Aeromonas↗
PubMed2001

Inhibition of a model protease--pyroglutamate aminopeptidase by a natural oligosaccharide gum from Hakea gibbosa.

The purpose of this study was to investigate the effect of the oligosaccharide gum from Hakea gibbosa on the activity of a model protease enzyme pyroglutamate aminopeptidase (5-oxoprolyl peptidase; EC 3.4.19.3) and to elucidate the mechanism responsible for the decreased activity. Enzyme kinetic studies were conducted at 37 degrees C in 100 mM potassium phosphate buffer with 10 mM EDTA, 5% (v/v) glycerol, and 5 mM DTT (pH 8) for 15 min and were performed both in the presence and absence of the gum. Enzymatic activity was determined by a colorimetric assay using the specific substrate L-pyroglutamic acid beta-napthylamide. The enzyme kinetics was studied at various substrate and gum concentrations. The velocity of the reaction was determined by the amount of the product (beta-napthylamine) liberated at each substrate and gum concentration. The Ks and Vmax of the enzyme in the absence of the gum were 24.40+/-2.14 microM and 502.95+/-28.90 nmoles x min(-1) x mg protein(-1), respectively. As the concentration of the gum was gradually increased from 0.1 to 2%, the value of the Vmax decreased from 318.94+/-21.46 to 158.83+/-24.51 nmoles x min(-1) x mg protein(-1) while Ks increased from 17.42+/-4.6 to 63.03+/-1.89 microM. The mechanism for the inhibition of the enzyme by Hakea appeared to be a mixed-linear type (a type of non-competitive inhibition) as suggested from Hanes-Woolf, Dixon and Cornish-Bowden plots. The turnover number, kcat, calculated for the enzyme also decreased from 14.09+/-0.81 to 4.45+/-0.69 min(-1) as the concentration of the inhibitor was incrementally increased from 0 to 2% (w/v). The K(i) and alphaK(i) calculated from Dixon and Cornish-Bowden plots were found to be 0.31+/-0.11% (w/v) and 1.33+/-0.42% (w/v), respectively. The natural gum from Hakea gibbosa was effective in non-competitively inhibiting the enzyme pyroglutamate aminopeptidase. Thus, the natural gum may be a promising additive not only for its sustained-release and mucoadhesive properties as shown previously, but also for its ability to slow the enzymatic degradation of therapeutic polypeptides incorporated in dosage forms.

Hydrogen-Ion Concentration↗
PubMed2003

Demonstration of puromycin-sensitive alanyl aminopeptidase in Alzheimer disease brain.

Puromycin-sensitive alanyl aminopeptidase (PSA, EC 3.4.11.14) is a member of the ubiquitous aminopeptidase family, which cleaves N-terminal amino acids from proteins. PSA is suggested to function as a trimming protease in the MHC class I pathway, which is activated in brains of Alzheimer disease (AD). We examined the immunohistochemical localization of PSA in brains of AD and control cases using a rabbit anti-PSA. In the control cases, the antiserum revealed staining in a few glial cells and blood vessels. In AD brain, however, intensely stained cells were found richly in the cerebral cortex. Double immunofluorescence studies confirmed that PSA-positive cells were reactive microglia. Such PSA-positive reactive microglia tended to locate in and around senile plaques and were sometimes observed to associate with neurons containing neurofibillary tangles. The present result indicates that reactive microglia express PSA-immunoreactive molecules, probably in association with the pathological conditions of AD.

Alzheimer Disease↗
PubMed1986

Purification and characterization of an enkephalin-degrading dipeptidyl-aminopeptidase from porcine brain.

A porcine brain dipeptidyl-aminopeptidase (DAP) has been purified more than 2400-fold from a crude mitochondrial fraction containing synaptosomes. This enzyme catalyzes the release of free Tyr-Gly from Leu-enkephalin (Km = 2.5 microM) with an optimal activity between pH 6.0 and pH 8.0. The enzyme appears homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis devoid of detectable contaminating aminopeptidase activities. The native enzyme is a monomeric protein with a molecular weight of 51,000 +/- 1,000 and an isoelectric point of 4.6 +/- 0.1. This enzyme cosediments with synaptosomes on a Ficoll-sucrose gradient and is partially associated with synaptic plasma membranes. Its activity is inhibited by the metal-chelating agents ethylenediaminetetraacetate and o-phenanthroline. It is not inhibited by the OH-reactive agent phenylmethanesulfonyl fluoride and SH-reactive agents such as p-(chloromercuri)benzoate and N-ethylmaleimide. Among the various biologically active peptides tested, the purified enzyme releases efficiently the N-terminal dipeptide moiety from enkephalins, Trp-Met-Asp-Phe-NH2 (CCK4), and Gly-Trp-Met-Asp-Phe-NH2 (CCK5). At variance, the native peptides CCK8, substance P, neurotensin, and angiotensin II are not cleaved by the DAP. This enzyme is different from other unspecific DAPs, as well as from enkephalin-degrading DAPs previously reported, by its molecular weight and substrate specificity.

Animals↗
PubMed1997

Development and in vitro evaluation of systems to protect peptide drugs from aminopeptidase N.

PURPOSE: Develop and evaluate systems to prevent aminopeptidase N caused enzymatic degradation of perorally administrated peptide drugs. METHODS: Bacitracin was covalently bound to the unabsorbable carrier matrix poly(acrylic acid) (paa) in order to avoid any dilution effects of the inhibitor in the intestine as well as systemic toxic side effects. The inhibitory effect of this conjugate, of neutralized paa and N-acetylcysteine was evaluated using a brush border membrane model. RESULTS: Whereas within 6 h of incubation 65.3 +/- 3.7 mumol/l of the substrate (L-leucine p-nitroanilide) was hydrolyzed under our assay conditions, this metabolism was reduced to 44.5 +/- 6.3 mumol/l and 49.0 +/- 8.8 mumol/l (n = 3-5; +/- S.D.) using 1.5% bacitracin-polymer conjugate and 0.5% N-acetylcysteine, respectively. The same amount of bacitracin as immobilized to the polymer exhibited a comparably weaker inhibitory effect. Neutralized paa did not inhibit membrane bound aminopeptidase N. Covering the membrane with a thin mucus layer led to a significantly lowered inhibitory effect of all tested agents. CONCLUSIONS: The immobilization of enzyme inhibitors to a carrier matrix and the use of N-acetylcysteine as a novel inhibitor are promising strategies in order to overcome the enzymatic barrier caused by membrane bound peptidases. However the use of effective mucolytic agents seems to be a prerequisite.

Acetylcysteine↗
PubMed1993

Processing of chimeric mammalian cytochrome b5 precursors in Escherichia coli: reaction specificity of signal peptidase and identification of an aminopeptidase in post-translocational processing.

A chimeric precursor interlinked by an arginine residue between the full-length signal sequence of alkaline phosphatase and the eukaryotic cytoplasmic cytochrome b5 was constructed. Expression of the chimeric precursor protein in Escherichia coli resulted in efficient export of spectrally authentic cytochrome b5 into the periplasm [Karim, Harding, Evans, Kaderbhai and Kaderbhai (1993) Bio/Technology 11, 612-618]. On sequencing, the apparent absence of arginine at the N-terminus of the secreted cytochrome b5 implied that the chimera was either miscleaved by signal peptidase or further processed following signal excision by an uncharacterized peptidase. The influence of the N-terminal region of cytochrome b5 on the unusual processing of the chimeric precursor was investigated by engineering a number of variant forms in which the region between Arg+1 and the mature portion of cytochrome b5 was extended and varied. Observations of the in vivo processed patterns of these variant cytochrome b5 forms exported into the periplasm revealed that the absence of arginine was due to neither miscleavage of the translocated precursor by the signal peptidase nor the nature of the early region of cytochrome b5. In fact, the selective excision of the arginine residue occurred subsequent to signal sequence deletion by an aminopeptidase which was sensitive to the metal chelator o-phenanthroline. We show that this aminopeptidase also participates in the trimming of the N-terminal arginine residue of the bacterial alkaline phosphatase to generate the three isoenzymes in the periplasm.

Amino Acid Sequence↗
PubMed1994

Dipeptidyl aminopeptidase III of guinea-pig brain: specificity for short oligopeptide sequences.

A dipeptidyl aminopeptidase III-type activity has been purified from the cytoplasm of guinea-pig brain using arginyl-arginyl-7-amido-4 methylcoumarin as substrate. The enzyme was purified 754-fold relative to the crude homogenate and with a 12.7% recovery. The purified enzyme was found to have a relative molecular weight of 85,000 and consists of one polypeptide chain of relative molecular weight 80,000, on the basis of its migration on calibrated sodium dodecyl sulphate-polyacrylamide gel electrophoresis gel. It is highly sensitive to the presence of chelating agents, sulphydryl reactive agents, and the dipeptide Tyr-Tyr. Dithiothreitol (1 mM) reduced activity by 28%, and 36 and 65% inhibition was noted with phenylmethylsulphonyl fluoride and puromycin (both at 1 mM), respectively. Little or no inhibition was observed with bestatin, bacitracin, captopril, amastatin, and arphamenine B. The purified enzyme released dipeptide moieties from a wide range of peptides including enkephalin sequences and also angiotensin sequences up to the octapeptide angiotensin II. These sequences inhibited the hydrolysis of arginyl-arginyl-7-amido-4-methylcoumarin by dipeptidyl aminopeptidase III with Ki values in the micromolar range. No hydrolysis was observed with angiotensin I or with peptide sequences containing more than 10 amino acids. No hydrolysis was observed also with peptide sequences containing a Pro residue on either side of the sissile bond. Peptides containing less than four amino acids were not hydrolysed.

Amino Acid Sequence↗
PubMed1998

Betulinic acid inhibits aminopeptidase N activity.

The triterpene betulinic acid inhibits the activity of aminopeptidase N (EC 3.4.11.2) in a dose-dependent manner. An IC50 of 7.3 +/- 1.4 microM was determined for betulinic acid. This inhibitory activity is higher than that of bestatin' (IC50 = 16.9 +/- 4.1 microM), a well known inhibitor of this enzyme. The finding supports the idea that betulinic acid acts as anti-melanoma agent via inhibition of aminopeptidase N activity.

Adjuvants, Immunologic↗
PubMed2006

Structure of aminopeptidase N from Escherichia coli suggests a compartmentalized, gated active site.

Aminopeptidase N from Escherichia coli is a major metalloprotease that participates in the controlled hydrolysis of peptides in the proteolytic pathway. Determination of the 870-aa structure reveals that it has four domains similar to the tricorn-interacting factor F3. The thermolysin-like active site is enclosed within a large cavity with a volume of 2,200 A(3), which is inaccessible to substrates except for a small opening of approximately 8-10 A. The substrate-based inhibitor bestatin binds to the protein with minimal changes, suggesting that this is the active form of the enzyme. The previously described structure of F3 had three distinct conformations that were described as "closed," "intermediate," and "open." The structure of aminopeptidase N from E. coli, however, is substantially more closed than any of these. Taken together, the results suggest that these proteases, which are involved in intracellular peptide degradation, prevent inadvertent hydrolysis of inappropriate substrates by enclosing the active site within a large cavity. There is also some evidence that the open form of the enzyme, which admits substrates, remains inactive until it adopts the closed form.

Amino Acid Sequence↗
PubMed2002

Silencing of midgut aminopeptidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiensis toxin receptor.

Insecticidal crystal proteins of Bacillus thuringiensis bind to receptors in the midgut of susceptible insects leading to pore formation and death of the insect. The identity of the receptor is not clearly established. Recently a direct interaction between a cloned and heterologously expressed aminopeptidase (slapn) from Spodoptera litura and the Cry1C protein was demonstrated by immunofluorescence and in vitro ligand blot interaction. Here we show that administration of slapn double-stranded RNA to S. litura larvae reduces its expression. As a consequence of the reduced expression, a corresponding decrease in the sensitivity of these larvae to Cry1C toxin was observed. The gene silencing was retained during the insect's moulting and development and transmitted to the subsequent generation albeit with a reduced effect. These results directly implicate larval midgut aminopeptidase N as receptor for Bacillus thuringiensis insecticidal proteins.

Animals↗
PubMed2007

Knockdown of aminopeptidase-N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein Cry1Ac.

Aminopeptidase-N (APN) and cadherin proteins located at the midgut epithelium of Helicoverpa armigera have been implicated as receptors for the Cry1A subfamily of insecticidal proteins of Bacillus thuringiensis. Ligand blot analysis with heterologously expressed and purified H. armigera Bt receptor with three closely related Cry1A proteins tentatively identified HaAPN1 as an interacting ligand. However, to date there is no direct evidence of APN being a functional receptor to Cry1Ac in H. armigera. Sf21 insect cells expressing HaAPN1 displayed aberrant cell morphology upon overlaying with Cry1Ac protein. Down-regulating expression of HaAPN1 by RNA interference using double-stranded RNA correlated with a corresponding reduction in the sensitivity of HaAPN1-expressing cells to Cry1Ac protein. This clearly establishes that insect cells expressing the receptor recruit sensitivity to the insecticidal protein Cry1Ac, and their susceptibility is directly dependent on the amount of HaAPN1 protein expressed. Most importantly, silencing of HaAPN1 in H. armigera in vivo by RNA interference resulted in reduced transcript levels and a corresponding decrease in the susceptibility of larvae to Cry1Ac. BIAcore analysis of HaAPN1/Cry1Ac interaction further established HaAPN1 as a ligand for Cry1Ac. This is the first functional demonstration of insect aminopeptidase-N of H. armigera being a receptor of Cry1Ac protein of B. thuringiensis.

Animals↗
PubMed2003

Differential regulation of aminopeptidase N (CD13) by transendothelial migration and cytokines on human eosinophils.

Aminopeptidase N (CD13) is a cell surface metalloprotease involved in growth regulation, tumor invasion, and down-regulation of regulatory peptides. CD13 expression on eosinophils in bronchoalveolar lavage (BAL) of asthmatics 10 minutes and 18 hours after segmental allergen provocation was significantly increased (+225% to +294%) compared to blood eosinophils. In vitro CD13 expression could be induced on blood eosinophils by transendothelial migration of the cells across interlenkin (IL) 1beta-activated human umbilical cord vein endothelial cells (HUVECs) as well as by the exposure to the cytokines IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF). The cytokines GM-CSF and IL-5 were significantly less effective in inducing CD13 compared to IL-3. The IL-3-induced expression of CD13 was decreased in the presence of the protein-synthesis inhibitor cycloheximide (-8.8%). Moreover, blocking of CD13 by the protease inhibitors actinonin and bestatin significantly enhanced migration (+40.0% to +80.0%) of eosinophils across HUVEC monolayers. In summary, the data suggest that CD13 is regulated both by the process of transmigration and by the cytokine IL-3. Further, CD13 itself seems to be involved in the process of eosinophil transmigration. aminopeptidase Nendothelial cellseosinophilsinterleukin-3interleukin-5transendothelial migration

Adult↗
PubMed1993

Endopeptidase activity of cathepsin C, dipeptidyl aminopeptidase I, from bovine spleen.

By employing various synthetic substrates, as well as soluble denatured protein substrate (TAP-lysozyme) and its derivatives, endopeptidase activity of cathepsin C, dipeptidyl aminopeptidase I [EC 3.4.14.1], from bovine spleen was investigated. Cathepsin C efficiently degraded Z-Phe-Arg-MCA, Pro-Phe-Arg-MCA, and Suc-Leu-Leu-Val-Tyr-MCA. This endopeptidase activity required sulfhydryl reagents and halide ions, as in the case of the dipeptidyl aminopeptidase (DAP) activity. We confirmed that this endopeptidase activity is due to cathepsin C itself based on the results on gel-filtration and anion-exchange chromatographies, comparative studies of the inhibitory effects of leupeptin and E-64 on this activity and those of cathepsins B and L, and further the competitive inhibitions by mutual substrates for the DAP and endopeptidase activities of cathepsin C. We also found that cathepsin C endopeptidase activity towards TAP-lysozyme and its N-alpha-acetylated tryptic peptides showed marked dependence on sulfhydryl reagents and chloride ion. Thus, we concluded that cathepsin C has endopeptidase activity as well as DAP activity. The binding energy between the enzyme and the amino acid side chains of the substrate may be as important for the endopeptidase activity as is the electrostatic interaction between the enzyme and the free alpha-amino group of the substrate for the DAP activity.

Animals↗