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PubMed1996

Cloning and genetic analysis of the Vibrio cholerae aminopeptidase gene.

The structural gene for the Vibrio cholerae leucine aminopeptidase (lap) was cloned and sequenced. The cloned DNA fragment contained a 1,503-bp open reading frame potentially encoding a 501-amino-acid polypeptide with a calculated molecular mass of 54,442 Da. The deduced amino acid sequence of the entire protein showed high homology with the sequence of Vibrio proteolyticus leucine aminopeptidase. The residues potentially involved in binding the zinc ions were completely conserved in the V. cholerae aminopeptidase as well as in the V. proteolyticus aminopeptidase. The recombinant protein was partially purified and characterized. The molecular mass was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 34 kDa, suggesting a processing of the protein to acquire the mature form. The protease showed maximum activity at pH 9.0 and was thermostable at 70 degrees C. The substrate leucyl-p-nitroanilide was cleaved by the protease, and its activity was inhibited by EDTA and bestatin. These results suggested that the protein was a leucine aminopeptidase. The PCR analysis of lap gene distribution showed that it was widely distributed among the V. cholerae strains. It was not present in the other species examined.

Amino Acid Sequence↗

PubMed1982

Use of arginine aminopeptidase activity in characterization of arginine-utilizing mycoplasmas.

The aminopeptidase activity of arginine-utilizing mycoplasmas was investigated with 20 aminoacyl beta-naphthylamide substrates. High levels of arginyl-beta-naphthylamide hydrolysis were demonstrated in 6 of 11 species when extracts of concentrated washed organisms were used. Relatively low arginine aminopeptidase activity was demonstrated with similar extracts from 22 species not utilizing arginine. The high level of arginine aminopeptidase activity could also be demonstrated with unwashed, unconcentrated samples of the same 6 species and also with Mycoplasma arthritidis. The procedure for preparing the extract of M. arthritidis appeared to remove the arginine aminopeptidase activity which was demonstrated to be present in the untreated culture. Fluorogenic and chromogenic tests were developed whereby this distinctive arginine aminopeptidase activity could be demonstrated within 4 h with the use of small volumes of broth culture (10 microliter) or single colonies, thus providing a rapid test for early characterization of some Mycoplasma species.

Aminopeptidases↗

PubMed1989

Leucine aminopeptidase M-induced reductions in blood pressure in spontaneously hypertensive rats.

Leucine aminopeptidase M significantly reduced blood pressure for up to 40 minutes when infused intracerebroventricularly into anesthetized spontaneously hypertensive rats (SHR) from a mean +/- SEM of 190 +/- 4 to 94 +/- 7 mm Hg and also in normotensive Wistar-Kyoto (WKY) rats from 138 +/- 5 to 68 +/- 8 mm Hg. Cerebrospinal fluid levels of angiotensin II (Ang II) and III were measured by radioimmunoassay and indicated drops with leucine aminopeptidase M infusion in SHR (from 36 +/- 6 to 11 +/- 1 pg/100 microliters) and in WKY rats (from 33 +/- 9 to 13 +/- 1 pg/100 microliters). Pretreatment with the specific angiotensin receptor antagonist [Sar1, Thr8]Ang II (sarthran) significantly diminished the subsequent leucine aminopeptidase M-induced decreases in blood pressure in SHR and facilitated recovery to base level blood pressure and heart rate in blood strains. Thus, exogenous application of leucine aminopeptidase M into the brain lateral ventricles of SHR is temporarily effective at reducing blood pressure, and this effect appears dependent on the brain angiotensinergic system. This treatment also reduced blood pressure in WKY rats; however, pretreatment with sarthran was reasonably ineffective at preventing subsequent leucine aminopeptidase M-induced decreases in blood pressure.

Angiotensin II↗

PubMed1992

Differential expression of aminopeptidase-N on human ovarian granulosa and theca cells.

The expression of aminopeptidase-N and neutral endopeptidase in human ovarian tissue was examined using specific monoclonal antibodies for each of these peptidases and histochemical staining for enzyme activity. Aminopeptidase-N is a membrane-bound metalloprotease catalyzing the removal of N-terminal amino acids from peptides and was detected by immunofluorescence staining on theca interna cells in secondary follicles and on luteinized thecal cells in preovulatory follicles and corpora lutea. However, aminopeptidase-N was not detected on granulosa cells. Peptidase activity was also detected by histochemical staining on theca interna cells and luteinized thecal cells. Luteinized granulosa cells showed peptidase activity, despite the lack of aminopeptidase-N. Neutral endopeptidase was not detected in ovarian granulosa and thecal cells. These observations indicate that aminopeptidase-N can be a useful surface marker for thecal cells.

Adult↗

PubMed1998

Aminopeptidase B: a processing enzyme secreted and associated with the plasma membrane of rat pheochromocytoma (PC12) cells.

Aminopeptidase B (Ap-B) is a Zn2+-dependent exopeptidase which selectively removes Arg and/or Lys residues from the N terminus of several peptide substrates. Isolated and characterized from rat testes, this ubiquitous enzyme may participate in the final stages of precursor processing mechanisms. To test this hypothesis, we have investigated the secretion and subcellular localization of this enzyme in a rat cell line of pheochromocytoma (PC12 cells). By using a combination of biochemical and immunocytochemical methods, the following observations were made: (i) the level of aminopeptidase B detectable in the cell culture medium increased with time; (ii) 8-bromo-adenosine 3'-5'-cyclic monophosphate and the Ca2+ ionophore A23187 both stimulated enzyme liberation in the culture medium; (iii) brefeldin A, an inhibitor of vesicular transport from the endoplasmic reticulum to the Golgi apparatus, decreased enzyme secretion in a time-dependent manner; (iv) whereas nocodazole, a microtubule depolymerizing agent, inhibited enzyme secretion, cytochalasin D, a microfilament disruption agent, had no effect on released aminopeptidase B level; (v) immunofluorescence demonstrated the presence of aminopeptidase B in the Golgi apparatus; (vi) immunofluorescence, electron microscopy and tests of enzyme activity on intact cells showed an association of the peptidase with the external face of the plasma membrane. Together these data strongly argued in favour of the enzyme secretion by PC12 cells. It is concluded that aminopeptidase B may participate in processing events occurring either during its intracellular transport along the secretory pathway or at the plasma membrane level, or both.

Aminopeptidases↗

PubMed1986

Uptake of 450191-S, a 1H-1,2,4-triazolyl benzophenone derivative, in the everted sac of rat small intestine: role of intestinal aminopeptidases.

5-[(2-Aminoacetamido)methyl]-1-[p-chloro-2-(o-chlorobenzoyl)phenyl ]-N,N-dimethyl-1 H-1,2,4-triazole-3-carboxamide hydrochloride dihydrate (450191-S), a sleep inducer, is a ring-opened derivative of 1,4-benzodiazepine and has been reported to be activated by intestinal aminopeptidases in a step of absorption. In this study, we investigated the role of the intestinal aminopeptidases in the uptake of 450191-S by the intestine by using various aminopeptidase inhibitors in everted sacs of rat small intestine in vitro. Glycylglycine did not affect the desglycylation of 450191-S but glycyl-L-leucine or L-leucyl-L-leucine inhibited the reaction. This inhibition was accompanied by reduced concentration of 450191-S metabolites in the intestinal tissue. When the incubation was carried out at 0 degrees C or with puromycin, the desglycylation was also inhibited and the concentration was similarly reduced. Since the extent of inhibition of the desglycylation of 450191-S was negatively correlated with the total concentration of 450191-S metabolites in the intestinal tissue (r = 0.9660), the aminopeptidases must play an important role in the uptake of 450191-S by the intestine. To confirm this, we examined the uptake of the desglycylated product of 450191-S, 8-chloro-6-(2-chlorophenyl)-N,N-dimethyl-4H-1,2,4-triazolo- [1,5-alpha] [1,4]-benzodiazepine-2-carboxamide (M-1) in the same manner. No effect of dipeptides, puromycin or reduced temperature was found on M-1 uptake by the intestine, leading to the conclusion that the aminopeptidases are important for enhancing the uptake of 450191-S in the intestine.

Aminopeptidases↗

PubMed2004

Distribution of adipocyte-derived leucine aminopeptidase (A-LAP)/ER-aminopeptidase (ERAP)-1 in human uterine endometrium.

Adipocyte-derived leucine aminopeptidase (A-LAP, endoplasmic reticulum aminopeptidase ERAP1) is specialized to produce peptides presented on the class I major histocompatibility complex (MHC) by trimming epitopes to eight or nine residues, in addition to its enzymatic activity to degrade angiotensin II. Previously we identified placental leucine aminopeptidase (P-LAP), another member of the oxytocinase subfamily of aminopeptidases, in human uterine endometrial epithelial cells. Here we analyzed the distribution of A-LAP in human cyclic endometrium. Western blotting analysis showed that A-LAP was present in the endometrial tissue throughout the menstrual cycle. Immunohistochemical (IHC) analysis of A-LAP showed a similar distribution to that of P-LAP. A-LAP was localized predominantly in the endometrial glands and the luminal surface epithelium. However, the intracellular localization change that accompanied apocrine secretion, which was observed in P-LAP, was not shown in A-LAP. Subcellular localization of A-LAP, demonstrated by immunofluorescence, was ER in the cultured glandular epithelial cells. Our results indicate that A-LAP may fit the endometrial localization as an antigen-presenting ER aminopeptidase. Further understanding of the function(s) of A-LAP in the endometrium appear likely to yield insights into the cyclic changes during the normal endometrial cycle.

Adult↗

PubMed2003

Identification of cytosolic leucyl aminopeptidase (EC 3.4.11.1) as the major cysteinylglycine-hydrolysing activity in rat liver.

Cysteinylglycine hydrolysis is a step in the metabolism of glutathione and glutathione S-conjugates. We had previously observed that in rat liver the enzymatic activity is predominantly located in the cytosol. Here we demonstrate that cytosolic leucyl aminopeptidase (EC 3.4.11.1) is the major cysteinylglycine hydrolysing activity in rat liver. Evidence was obtained from the use of peptidase inhibitors and from immunoprecipitation studies using Pansorbin-coupled antibodies raised against hog kidney cytosolic leucyl aminopeptidase. Both isolated cytosolic leucyl aminopeptidase and the cysteinylglycine-hydrolysing activity in rat liver cytosol are bound with equal efficiency to the affinity matrix. We demonstrate that cytosolic leucyl aminopeptidase exhibits leucinamidase and cysteinylglycinase activity. Cysteinylglycine, cystinyl-bis-glycine, S-nitrosocysteinylglycine, and bimane-S-cysteinylglycine are hydrolysed at high rates; low activity is seen with leukotriene D4. Our findings establish a previously unrecognised physiological function of cytosolic leucyl aminopeptidase, participating in glutathione metabolism and in the degradation of glutathione S-conjugates via the mercapturic acid pathway.

Acetylcysteine↗

PubMed2003

Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets.

Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanyl-aminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+ CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAP-specific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.

Alanine Transaminase↗

PubMedDate not supplied

Comparative studies on the dodecameric and hexameric forms of yeast aminopeptidase I.

Yeast aminopeptidase I, when purified from autolysates of brewer's yeast, is obtained in two molecular froms a) the enzymatically active dodecameric complex (Mr = 640,000, s20,w = 22 S) and b) inactive hexamers (Mr = 320,000, s20,w = 12 S). Although the amino acid composition of the 12 S protein is very similar to that of the active enzyme,the hexamers behave differently in ionic exchange chromatography and during electrophoresis on polyacrylamide gels. Moreover, the antigenic properties of 12 S and 22 S aminopeptidase forms suggest a considerable degree of structural diversity. Several strains of Saccharomyces cerevisiae did not contain hexameric forms although their 22 S aminopeptidase was immunologically indistinguishable from brewer's yeast aminopeptidase. It is proposed that the hexameric protein is the result of "unproductive" aggregation of aminopeptidase subunits.

Amino Acids↗

PubMed2002

Characterization of aminopeptidase activity from three species of microsporidia: Encephalitozoon cuniculi, Encephalitozoon hellem, and Vittaforma corneae.

Microsporidia are obligate intracellular parasites of the phylum Microspora. To date, more than 1,200 species within 144 genera have been described, with 14 infecting humans. Currently, no effective treatment exists for human microsporidiosis. In this study, the biochemical properties of the aminopeptidases were investigated within several species of microsporidia. Aminopeptidase activity was detected in 3 species of microsporidia, Encephalitozoon cuniculi, E. hellem, and Vittaforma corneae, using a fluorometric substrate assay. Each species exhibited distinct aminopeptidase properties. The cytosolic neutral aminopeptidase activities of the Encephalitozoon spp. were characterized as preferentially cleaving leucine, whereas those of V. corneae cleaved arginine. Native polyacrylamide gel electrophoresis estimated the molecular mass of E. cuniculi, E. hellem, and V. corneae as 74, 72, and 79 kDa, respectively. Enzymatic activity was inhibited by bestatin and it's analogue, nitrobestatin, indicating that the enzyme was an aminopeptidase for all species. Inhibition with the chelating agents ethylenediaminetetraacetic acid and 1,10phenanthroline characterized the enzymes as metalloaminopeptidases. Subcellular fractionation of the 3 microsporidial species suggested that the enzyme activity was localized in the cytosolic fraction. Optimal enzyme activity was observed at pH 7.2 for all species. This is the first report of enzyme characterization from these 3 species of microsporidia.

Aminopeptidases↗

PubMed1975

A biochemical study of aminopeptidases in the palatal mucosa of the rat following tooth extractions.

Aminopeptidase activities were studied biochemically from the supernatants of the homogenates prepared from the normal and post extraction palatal mucosa of the rat. The determinations were carried out at 0.5, 1, 2, 4, 8 and 16 hours and 1, 2, 3, 5 and 7 days following extraction using N-L-aminoacyl-2-naphthylamines of arginine, glycine, leucine, proline and valine as the substrates. In addition, in the second part of the study the hydrolysis of N-L-aminoacyl-2-naphthylamines of alanine, histidine, isoleucine, lysine, phenylalanine, serine, threonine, tyrosine and valine was tested at 1, 3 and 5 days following extraction. The effect of chloride ions (0.2 M NaCl) on the hydrolysis of arginine and lysine derivatives was also tested. The specific activities of aminopeptidases obtained from the extraction-wound side were compared to those of the control side at each time interval. Tooth extraction caused a considerable reduction in the specific activities of aminopeptidases in the surrounding palatal mucosa immediately after extraction (at 0.5 hr.). The specific activities in the wound tissue remained generally below the control levels for 24 hours and reached their maximum within 5 days following extractions. The highest relative increase was obtained with N-L-valyl-2-naphthylamine. Arginine aminopeptidase was activated by chloride ions on an average 20 per cent more in the wound tissue than the control. This effect can be considered suggestive of the activation of aminopeptidase B in the post extraction palatal mucosa.

Aminopeptidases↗

PubMed1986

Aminopeptidase III activity in normal and cataractous lenses.

Aminopeptidase III activity was demonstrated in extracts from several different mammalian lenses by the hydrolysis of Arg-MCA at pH 6.0. No more than a two-fold difference was seen in overall specific activity. Sections of bovine lenses were removed from the periphery to the center and assayed. A sharp decline in activity was observed in the inner cortical region, and little or no activity was observed in the lens nucleus. This correlated with an increase in the presence of low molecular weight peptides as determined by SDS polyacrylamide gel electrophoresis. The properties of the aminopeptidase from human lens tissue were the same as those previously reported for the purified enzyme from bovine lens. The aminopeptidase activity of normal and cataractous lenses was compared using 4 different substrates. The cataractous lenses had significantly less total aminopeptidase activity. However, little difference in specific activity was observed based on soluble lens protein content. Similarly, electrophoretic separations of normal and cataractous soluble proteins showed little or no differences in the content of low molecular weight peptides. Therefore, this major human lens aminopeptidase remains functional in the cataractous state.

Aging↗

PubMed1993

Cholestyramine and bile diversion lower the aminopeptidase activity in the intestinal brush border membrane of rats.

To examine the effect of bile acids on the activity of intestinal aminopeptidase in vivo, we measured the activity of aminopeptidase in the intestinal mucosa from rats fed the diet containing cholestyramine which sequesters luminal bile acids (experiment 1) and from bile diverted rats (experiment 2). After 32 h fasting, rats were refed for 16 h either of a standard diet (25% casein diets), the same diet containing cholestyramine, or the fat-free diet in experiment 1. In the intestinal washing, the content of total bile acids was markedly decreased with feeding cholestyramine and activities of trypsin and chymotrypsin were also lowered with cholestyramine. Cholestyramine feeding decreased the specific activity of aminopeptidase in the homogenate of intestinal mucosa but increased the specific activities of sucrase and alkaline phosphatase. All these parameters were not modified by the fat-free diet. In experiment 2, bile diverted and sham operated rats were refed the standard diet for 16 h with prior 32 h fasting. Bile diversion, like cholestyramine feeding, lowered the content of total bile acids, the activities of pancreatic hydrolases in the intestinal washings, and the specific activity of aminopeptidase in the intestinal mucosa. The specific activity of sucrase in the intestinal mucosa was higher in bile diverted rats but the activity of alkaline phosphatase was not changed. These data indicate that the decreased abundance of intraluminal bile acid affects the activity of intestinal aminopeptidase not through the decreased absorption of dietary lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkaline Phosphatase↗

PubMed2006

Expression of endoplasmic reticulum aminopeptidases in EBV-B cell lines from healthy donors and in leukemia/lymphoma, carcinoma, and melanoma cell lines.

Peptide trimming in the endoplasmic reticulum (ER), the final step required for the generation of most HLA class I-binding peptides, implicates the concerted action of two aminopeptidases, ERAP1 and ERAP2. Because defects in the expression of these peptidases could lead to aberrant surface HLA class I expression in tumor cells, we quantitatively assayed 14 EBV-B cell lines and 35 human tumor cell lines of various lineages for: 1) expression and enzymatic activities of ERAP1 and ERAP2; 2) ER peptide-trimming activity in microsomes; 3) expression of HLA class I H chains and TAP1; and 4) surface HLA class I expression. ERAP1 and ERAP2 expression was detectable in all of the EBV-B and tumor cell lines, but in the latter it was extremely variable, sometimes barely detectable, and not coordinated. The expression of the two aminopeptidases corresponded well to the respective enzymatic activities in most cell lines. A peptide-trimming assay in microsomes revealed additional enzymatic activities, presumably contributed by other unidentified aminopeptidases sharing substrate specificity with ERAP2. Interestingly, surface HLA class I expression showed significant correlation with ERAP1 activity, but not with the activity of either ERAP2 or other unidentified aminopeptidases. Transfection with ERAP1 or ERAP2 of two tumor cell lines selected for simultaneous low expression of the two aminopeptidases resulted in the expected, moderate increases of class I surface expression. Thus, low and/or imbalanced expression of ERAP1 and probably ERAP2 may cause improper Ag processing and favor tumor escape from the immune surveillance.

Aminopeptidases↗

PubMed1999

Proteolytic enzymes in human leukemic lymphoid cells. III. Aminopeptidases, angiotensin-converting enzyme, and its inhibitor in cells of different immunological phenotype.

Activities of plasma membrane proteinases such as angiotensin-converting enzyme (ACE), aminopeptidases, and dipeptidyl peptidase IV (DPP-IV) were determined in lymphoid cells of various immunological phenotype which were obtained from 30 patients with lymphoproliferative diseases. The enzyme activities significantly varied depending on the immunological phenotype and stage of cell differentiation, but no correlation was found between activities of ACE, DPP-IV, and aminopeptidases in the cells of different type. The cell lysates studied contained at least two classes of aminopeptidases: metal- and sulfhydryl-dependent enzymes. A sulfhydryl-dependent aminopeptidase with activity optimum at pH 8. 5-9.0 was found for the first time and is suggested to be from a poorly studied aminopeptidase family. In addition to ACE, lysates of leukemic T- and B-cells were found to contain an inhibitor of ACE which was not previously described for these cells.

Aminopeptidases↗

PubMed2001

Aminopeptidase-like activities in Caenorhabditis elegans and the soybean cyst nematode, Heterodera glycines.

Aminopeptidase-like activities in crude whole body extracts of the free-living nematode Caenorhabditis elegans and the plant parasitic soybean cyst nematode Heterodera glycines were examined. General characteristics including pH optima, heat lability, and inactivation of enzyme by organic solvent were the same for the two species. All developmental stages of H. glycines exhibited activity. In older females, activity was present primarily in the eggs. Affinity for the substrate L-alanine-4-nitroanilide was the same regardless of the stage examined, and was similar for the two species (m for C. elegans and m for H. glycines). Nearly all (>95%) of C. elegans aminopeptidase-like activity was present in the soluble fraction of the extract, while H. glycines activity was distributed between the soluble and membrane fractions. Specific activities of the soluble enzymes were highest in C. elegans and H. glycines juveniles. The C. elegans enzyme was susceptible to a number of aminopeptidase inhibitors, particularly to amastatin and leuhistin, each of which inhibited aminopeptidase-like activity more than 90% at 90 microm. In H. glycines, aminopeptidase-like activity was inhibited 39% by amastatin at 900 microm. The apparent molecular weight of the soluble C. elegans enzyme is 70-80 kDa. Some activity in H. glycines is present in the 70-80 kDa range, but most activity (80-90%) is associated with a very high molecular weight (>240 kDa) component.

Amino Acids↗

PubMed1992

Evidence for an apical sorting signal on the ectodomain of human aminopeptidase N.

In polarized epithelial cells aminopeptidase N is targeted to the apical membrane. The aim of this study was to determine whether a sorting signal is necessary for its correct transport to the apical membrane and, if so, to localize this sorting signal to one of the domains of the transmembrane protein. Anchor-minus aminopeptidase N, consisting of the hemagglutinin signal peptide including its cleavage site, and the ectoplasmic domain of human aminopeptidase N were stably expressed in Madin-Darby canine kidney cells cultured on polycarbonate filters. By measurement of the enzymatic activity it was found that the anchor-minus aminopeptidase N was secreted in a polarized manner to the apical side. As a reference the secretion of the secretory granule protein, cystatin C, was likewise studied. Cystatin C was found to be secreted in a nonpolarized manner to both domains. Our data thus show that human aminopeptidase N carries an apical sorting signal and that it is localized on the ectodomain of the enzyme.

Amino Acid Sequence↗