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PubMed1997

Secretion of an aminopeptidase during transition of third- to fourth-stage larvae of Ascaris suum.

Protease activity was identified in culture fluids collected during in vitro development of L3 to L4 larval stages of Ascaris suum. Fluorogenic peptide substrates with unblocked N-termini were specifically hydrolyzed indicating aminopeptidase activity; a terminal arginyl residue was preferred. Culture fluids did not hydrolyze fluorogenic peptide substrates with blocked N-termini (endopeptidase substrates). The aminopeptidase activity was inhibited by 1,10-phenanthroline (metalloprotease inhibitor) and by amastatin and bestatin (aminopeptidase inhibitors); AEBSF (serine protease inhibitor), Z-phe-ala-FMK and E-64 (cysteine protease inhibitors), and pepstatin A (aspartyl protease inhibitor) had little effect on activity. The apparent molecular weight of the aminopeptidase was estimated by sucrose density gradient centrifugation at 293 kDa. The aminopeptidase displayed an acidic isoelectric point of 4.7. The peak secretion of the aminopeptidase was temporally associated with molting and suggests a function for the protease in this complex process.

Aminopeptidases↗

PubMed1976

Aminopeptidase II from Bacillus stearothermophilus.

1. The low molecular weight aminopeptidase (aminopeptidase II) from Bacillus stearothermophilus cells grown at 50 degrees C was purified to a homogeneous state. 2. Molecular weight determination by sodium dodecyl sulfate gel electrophoresis resulted in a value of 46 000 for the subunits. A molecular weight of 80 000-100 000 has been reported for the native enzyme. We therefore conclude that aminopeptidase II is a dimeric enzyme. 3. The amino-terminal sequence, the amino acid analysis and the subunit molecular weight of aminopeptidase II show no relationship to the corresponding data of aminopeptidase I. 4. Aminopeptidase II binds two Co2+ per subunit. The dissociation constants of these ions determined by binding studies and by kinetic analysis agree within experimental error.

Amino Acids↗

PubMed2001

Interactions of Streptomyces griseus aminopeptidase with amino acid reaction products and their implications toward a catalytic mechanism.

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.

Amino Acids↗

PubMed1963

SERUM LACTIC DEHYDROGENASE, LEUCINE AMINOPEPTIDASE AND 5-NUCLEOTIDASE ACTIVITIES: OBSERVATION IN PATIENTS WITH CARCINOMA OF THE PANCREAS AND HEPATOBILIARY DISEASE.

Serum lactic dehydrogenase, leucine aminopeptidase, 5-nucleotidase and alkaline phosphatase activities were investigated in a number of diseases involving the hepatobiliary system.Leucine aminopeptidase was found to be a sensitive indicator of biliary obstruction, serum 5-nucleotidase slightly less sensitive, and alkaline phosphatase appreciably less sensitive. Leucine aminopeptidase and 5-nucleotidase activities were often increased by malignant infiltration of the liver and primary hepatic disease even in the absence of jaundice.Serum lactic dehydrogenase was frequently increased in primary hepatic disease and malignant disorders but was not apparently affected by bile duct obstruction per se. Thirty-five of 45 patients with proved malignancy had increased lactic dehydrogenase levels.The highest leucine aminopeptidase levels were encountered in carcinoma of the head of the pancreas. The frequent increase in both serum lactic dehydrogenase and leucine aminopeptidase activities in patients with carcinoma of the head of the pancreas suggests that these combined estimations are useful laboratory procedures in the diagnosis of malignant extrahepatic obstruction.

Aminopeptidases↗

PubMed2001

Thiolation of polycarbophil enhances its inhibition of intestinal brush border membrane bound aminopeptidase N.

The purpose of this study was to evaluate the potential of polycarbophil-cysteine conjugates (PCP-Cys) as an oral excipient to protect leucine enkephalin (leu-enkp) from enzymatic degradation by the intestinal mucosa. Cysteine was covalently linked to polycarbophil by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC). Inhibitory activity was tested towards isolated aminopeptidase N and excised intact pig intestinal mucosa, with native mucus. Aminopeptidase N activity was assayed spectrophotometrically using L-leucine p-nitroanilide (leu-pNA) as a synthetic substrate and against the model peptide drug leu-enkp, by high-performance liquid chromatography (HPLC). Free cysteine at 6.3 and 63 microM (pH 6) significantly (p < 0.05) inhibited aminopeptidase N activity, and PCP-Cys (0.25% w/v, pH 6) had a significantly (p < 0.05) greater inhibitory effect than PCP on the aminopeptidase N activity towards both substrates. PCP-Cys completely protected leu-enkp against aminopeptidase N activity over a 2-h incubation period, whereas 83 +/- 4 and 60 +/- 7% remained stable in the presence of PCP and buffer only, respectively. Leu-enkp in the absence and presence of PCP (0.25% w/v) at pH 6 was completely digested by the intact intestinal mucosa at the 60- and 90-min incubation time points, respectively, whereas in the presence of PCP-Cys (0.25% w/v, pH 6) 11 +/- 3.5% of leu-enkp remained at the 120-min time point. Thiolation of PCP increased the stability of leu-enkp against the enzymatic degradation by aminopeptidase N and the intact intestinal mucosa, identifying a promising new excipient for peroral delivery of peptides.

Acrylic Resins↗

PubMed1998

Inhibition of alanyl aminopeptidase induces MAP-kinase p42/ERK2 in the human T cell line KARPAS-299.

Inhibition of alanyl aminopeptidase (EC 3.4.11.2, aminopeptidase N, CD13) expression, or activity compromise cell proliferation in a number of cell systems [1, 2, 3, 4, 5, 6]. The underlying mechanisms and the molecular components involved have not been identified as yet. In this study we show that inhibition of alanyl aminopeptidase enzymatic activity decreases the proliferation rate of the CD13-positive T cell line Karpas-299. By using the ATLAS cDNA expression array (Clontech) we identified the p42/ERK2 MAP kinase as one downstream target of probestin, a potent inhibitor of alanyl aminopeptidase. Probestin and another specific aminopeptidase inhibitor, actinonin, in addition to their capability of inducing erk-2 mRNA levels, significantly increase p42 phosphorylation state. This is the first report on signal transduction components possibly mediating the growth-modulatory effects of alanyl aminopeptidase inhibitors.

CD13 Antigens↗

PubMed1999

Ontogeny of puromycin-sensitive and insensitive aminopeptidase activities in several subcellular fractions of the rat brain.

Puromycin-sensitive and insensitive aminopeptidase (aminopeptidase M) activities are measured in several subcellular fractions of the rat brain cortex and subcortex during the first postnatal month. Tyr-beta-naphthylamide has been used as substrate and 20 microM puromycin as selective inhibitor. We have found that puromycin-sensitive aminopeptidase activity increases twofold in the synaptosomal and mitochondrial fractions in the first 6-9 postnatal days, just during the period of axonal and dendritic growth. This enzyme also has significant age-related changes in the nuclear fraction. The developmental pattern is different, depending on the subcellular fraction analyzed. Significant developmental changes of puromycin-insensitive aminopeptidase (aminopeptidase M) are only found in the myelinic and microsomal fractions and they are less significant than those found in the puromycin-sensitive aminopeptidase. It has been suggested that these enzyme activities could be involved in processes of cell proliferation, differentiation, and maturation.

Analysis of Variance↗

PubMed2000

cDNAs of aminopeptidase-like protein genes from Plodia interpunctella strains with different susceptibilities to Bacillus thuringiensis toxins.

Aminopeptidase N has been reported to be a Bacillus thuringiensis (Bt) Cry1A toxin-binding protein in several lepidopteran insects. cDNAs of aminopeptidase-like proteins from both Bt-susceptible RC688s and Bt-resistant HD198r strains of the Indianmeal moth, Plodia interpunctella, were cloned and sequenced. They contain 3345 and 3358 nucleotides, respectively, and each has a 3048 bp open reading frame that encodes 1016 amino acids. Putative protein sequences include 10 potential glycosylation sites and a zinc metal binding site motif of HEXXH, which is typical of the active site of zinc-dependent metallopeptidases. Sequence analysis indicated that the deduced protein sequences are most similar to an aminopeptidase from Heliothis virescens with 62% sequence identity and highly similar to three other lepidopteran aminopeptidases from Plutella xylostella, Manduca sexta, Bombyx mori with sequence identities of 51-52%. Four nucleotide differences were observed in the open reading frames that translated into two amino acid differences in the putative protein sequences. Polymerase chain reaction (PCR) confirmed an aminopeptidase gene coding difference between RC688s and HD198r strains of P. interpunctella in the PCR amplification of a specific allele (PASA) using preferential primers designed from a single base substitution. The gene mutation for Asp185-->Glu185 was also confirmed in two additional Bt-resistant P. interpunctella strains. This mutation is located within a region homologous to the conserved Cry1Aa toxin binding regions from Bombyx mori and Plutella xylostella. The aminopeptidase-like mRNA expression levels in the Bt-resistant strain were slightly higher than those in the Bt-susceptible strain. The sequences reported in this paper have been deposited in the GenBank database (accession numbers AF034483 for susceptible strain RC688s and AF034484 for resistant strain HD198r).

Amino Acid Sequence↗

PubMed2005

Discovery of a dual-function peptide that combines aminopeptidase N inhibition and kinin B1 receptor antagonism.

Previous analyses support that aminopeptidase N is a major inactivation pathway for high-affinity peptide ligands of the human and rabbit forms of the kinin B(1) receptor (agonists or antagonists). In this study, we found that the high-affinity antagonist B-9958 (Lys-Lys-[Hyp(3), CpG(5), D-Tic(7), CpG(8)]des-Arg(9)-BK; des-Arg(9)-BK, des-arginine(9)-bradykinin) is an aminopeptidase N substrate based on its capacity to compete for the hydrolysis of the chromogenic substrate L-Ala-p-nitroanilide by membranes isolated from human or rabbit arterial smooth muscle cells, its inactivation in the presence of these membranes (radioreceptor assay) and on its intense potentiation by the aminopeptidase N inhibitor amastatin in the rabbit aorta contractility assay (gain of 0.84 units in the pA(2) scale). Analogs of B-9958 in which the N-terminal Lys residue was substituted by D-Lys or D-Arg (B-10352 and B-10356, respectively) showed reduced affinity at the human or rabbit B(1) receptors (1.2-2.8-fold), as estimated by the displacement of [(3)H]Lys-des-Arg(9)-BK binding, but were more potent antagonists of des-Arg(9)-BK-induced contraction of the rabbit aorta than B-9958 in the absence of amastatin; they were not potentiated by the latter inhibitor. Unexpectedly, B-10356 inhibited L-Ala- p-nitroanilide hydrolysis without being inactivated, suggesting that it is an aminopeptidase N inhibitor. This was verified because B-10356 (but not B-10352) potentiated peptides unrelated to kinins but susceptible to aminopeptidase N inactivation (angiotensin III, thrombin receptor hexapeptide agonist). B-10356 inhibits dual molecular targets (aminopeptidase N enzyme K(i), 0.9-2.2 microM; kinin B(1) receptor binding K(i), 0.5-1.5 nM), and this may be an advantage for specific therapeutic applications (e.g., inhibition of angiogenesis).

Animals↗

PubMed1987

Identification of the structural gene for dipeptidyl aminopeptidase yscV (DAP2) of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae lacking dipeptidyl aminopeptidase yscV were isolated from a strain already defective in dipeptidyl aminopeptidase yscIV, an enzyme with overlapping substrate specificity. The mutants were identified by a staining technique with the chromogenic substrate Ala-Pro-4-methoxy-beta-naphthylamide to screen colonies for the absence of the enzyme. One of the mutants had a thermolabile activity, indicating that it contained a structural gene mutation. The 53 mutants analyzed fell into one complementation group that corresponded to the yscV structural gene, DAP2. The defect segregated 2:2 in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Diploids heterozygous for DAP2 displayed gene dosage effects with respect to yscV enzyme activity. The absence of dipeptidyl aminopeptidase yscV or the combined loss of both dipeptidyl aminopeptidases yscIV and yscV did not affect mitotic growth under rich or poor growth conditions. In contrast to the dipeptidyl aminopeptidase yscIV lesion (ste13), which leads to alpha sterility because strains secrete incompletely processed forms of the alpha-factor pheromone, the dipeptidyl aminopeptidase yscV lesion did not affect mating, and strains produced fully active alpha-factor pheromone. dap2 mutants did not show any obvious phenotype under a variety of conditions tested.

Dipeptidyl-Peptidases and Tripeptidyl-Peptidases↗

PubMedDate not supplied

Alanyl-aminopeptidase activity decrease in cerebrospinal fluid of Alzheimer patients.

We analyzed the amino acid concentrations and aminopeptidase activities in plasma and the cerebrospinal fluid (CSF) of Alzheimer patients. Correlations were calculated between these parameters and the degree of cognitive impairment, duration of the disease and age. In comparison with a control group, no changes were found in the levels of amino acids in CSF or plasma of Alzheimer patients. Alanyl-aminopeptidase activity was significantly lower in the CSF of Alzheimer patients, whereas no differences in CSF were detected as regards the remaining aminopeptidases. The plasma/CSF ratio for aminopeptidase activities was higher in AD patients than in controls, although the difference was only significant for alanyl-aminopeptidase. Amino acid ratios did not show this general tendency. The correlations between plasma aspartate and glutamate concentrations and the stage of the disease, measured with the Mini Mental State Examination, were studied. Changes in aminopeptidase activities and their role in protein dysfunction underlying Alzheimer disease are discussed.

Aged↗

PubMed2005

Aminopeptidase N gene expression and abundance in caprine mammary gland is influenced by circulating plasma peptide.

This study examined the localization and the effect of circulating peptides on the expression of aminopeptidase N (EC 3.4.11.2) in caprine mammary gland. Four lactating goats in mid to late lactation were used in a crossover design and were subjected to 2 dietary treatments. Abomasal infusion of casein hydrolysate was used to increase the concentration of peptide-bound amino acid in the circulation. Samples of mammary gland tissue from each goat were taken by biopsy at the end of each treatment period to measure gene and protein expression of aminopeptidase N in the tissue. There were no measurable effects on feed intake and milk production for any of the treatments. Western blot analysis showed that aminopeptidase N is located on the basolateral side of parenchymal cells and not on the apical membranes. Abomasal infusion of casein hydrolysate caused a marked change in the profile of arterial blood free amino acids and peptide-bound amino acids smaller than 1500 Da. Abundance of aminopeptidase N mRNA and protein increased by 51 and 58%, respectively, in casein hydrolysate-infused goats compared with the control treatment. It was concluded that aminopeptidase N is one candidate actively involved in the mammary gland to support protein synthesis and milk production. In accordance with the nutritional conditions in the current experiment, it is suggested that aminopeptidase N expression is partly controlled by the metabolic requirements of the gland and postabsorptive forms of amino acids in the circulation.

Amino Acids↗

PubMed1997

Reduction of plasma glutamyl aminopeptidase activity in sporadic Alzheimer's disease.

Previously, we hypothesized that aminopeptidase-catalyzed proteolysis may limit the rate of beta-amyloid catabolism in brain and that reduction of a certain aminopeptidase activity may lead to deposition of peptidic metabolites represented by beta-amyloid and thus to Alzheimer's disease (AD). To explore this possibility in clinical situations and to seek a possible biochemical marker for the disease, we quantitated four classes of aminopeptidase activities in cerebrospinal fluids and heparinized plasma from sporadic AD patients and agematched controls collected in two independent medical institutions. We found that only plasma glutamyl aminopeptidase activity was significantly and consistently lower in AD patients. Although the mechanism leading to such a biochemical change in plasma remains to be elucidated, the results provide support for the aminopeptidase hypothesis and indicate that the enzyme activity may potentially be used as a diagnostic/ predictive marker for AD.

Aged↗

PubMed1990

Purification and properties of glutamyl aminopeptidase from chicken egg-white.

Hydrolytic activities characteristic for different aminopeptidases were detected in the egg-white of unfertilized chicken eggs, and one aminopeptidase was isolated in an electrophoretically homogeneous form. The isolated aminopeptidase preferentially hydrolyzed bonds of alpha-glutamyl residue at the NH(2)-end of synthetic substrates and peptides. The enzyme is a dimer with an M(r) of 320,000 and pI of 4.2. Its optimal pH and temperature are 7.6 and 60 degrees C, respectively. EDTA, amastatin, and N-bromosuccinimide are inhibitors, while Ca2++ and Mn2+ are activators of the enzyme Ca2+ also stabilizes the enzyme. According to the observed properties, the isolated chicken egg-white aminopeptidase belongs to the glutamyl aminopeptidases.

Aminopeptidases↗

PubMed2004

Aminopeptidase activity in the postmortem brain of human heroin addicts.

Several studies have reported that the chronic administration of opioids induces changes in the biosynthesis of endogenous opioid peptides or their precursors in specific brain regions of the adult central nervous system. However, little is known about the catabolic regulation of opioid peptides and its contribution to neuroadaptative changes underlying drug addiction. In the present study, we have analyzed the activity of two enkephalin-degrading enzymes (puromycin-sensitive aminopeptidase or PSA and aminopeptidase N or APN) and two functionally different, soluble aminopeptidases (aminopeptidase B and aspartyl-aminopeptidase) in postmortem samples of prefrontal cortex and caudate nucleus of eight human heroin addict brains and eight matched-controls. Enzyme activities were fluorimetrically measured using beta-naphthylamide derivatives. An increase in the activity of soluble PSA in the prefrontal cortex of heroin abusers was observed (heroin addict group: 51,452+/-3892 UAP/mg protein versus control group: 42,003+/-2597 UAP/mg protein; P<0.05), while the activity of the other peptidases in both brain regions remained unaltered. This result agrees with previous findings in morphine-tolerant rats, and indicates that soluble PSA may be involved in neurobiological processes which underlie heroin addiction.

Adult↗

PubMed2005

Subcellular distribution of membrane-bound aminopeptidases in the human and rat brain.

We evaluated the subcellular distribution of four membrane-bound aminopeptidases in the human and rat brain cortex. The particulate enzymes under study--puromycin-sensitive aminopeptidase (PSA), aminopeptidase N (APN), pyroglutamyl-peptidase I (PG I) and aspartyl-aminopeptidase (Asp-AP)--were fluorometrically measured using beta-naphthylamide derivatives. Membrane-bound aminopeptidase activity was found in all the studied subcellular fractions (myelinic, synaptosomal, mitochondrial, microsomal and nuclear fractions), although not homogenously. Human PSA showed highest activity in the microsomal fraction. APN was significantly higher in the nuclear fraction of both species, while PG I showed highest activity in the synaptosomal and myelinic fractions of the human and rat brain. The present results suggest that in addition to inactivating neuropeptides at the synaptic cleft, these enzymes may participate in other physiological processes. Moreover, these peptidases may play specific roles depending on their activity levels at the different subcellular structures where they are localized.

Aminopeptidases↗

PubMed1998

Sex differences and age-related changes in human serum aminopeptidase A activity.

Given that aminopeptidase A is primarily responsible for cleaving aspartic acid and converting angiotensin II to angiotensin III, the purpose of the present study was to evaluate the activity of aminopeptidase A by determination of glutamate aminopeptidase activity (GluAP) and aspartate aminopeptidase activity (AspAP) (reported respectively as aminopeptidase A and angiotensinase A activities) in human serum during development and ageing, in an apparently healthy population of 139 male and 148 female subjects. To measure GluAP and AspAP we used glutamyl- and aspartyl-2-naphthylamide as substrates. Significant age-related increases were observed in GluAP activity in males and females and in AspAP activity in females. In males, there were no age-related differences in AspAP activity. A significant correlation was observed between age and GluAP activity in the population analysed as a whole or according to sex. No correlation was demonstrated between age and AspAP activity either in the whole population or according to sex. These results may reflect the evolution of the functional status of susceptible circulating substrates during development and ageing.

Adolescent↗

PubMed1992

Relationship between the changes in placental blood flow resistance assessed by Doppler technique and maternal serum placental aminopeptidases, which degrade vaso-active peptides, in pre-eclampsia.

Our study showed that there were statistically significant correlations between the systolic and diastolic ratio (S/D) of maternal uterine or umbilical artery and the levels of maternal serum aminopeptidase activities in pre-eclampsia. Kininase I was positively correlated with the S/D ratios, whereas placental leucine aminopeptidase (P-LAP) and aminopeptidase A were negatively correlated with the S/D ratios. It is known that the increased S/D ratios reflect the increased utero-placental blood flow resistance. Since our previous study showed that placental aminopeptidases degrade vasoactive peptides such as oxytocin, angiotensin and bradykinin, which the fetus actively produces, our present study suggests that the increased vascular resistance in feto-placental circulation in pre-eclampsia is partly controlled by changes in vaso-active peptides, via degradation by placental aminopeptidases.

Adult↗