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Aminopeptidase
Kinds of Aminopeptidase Terms modified by Aminopeptidase Selected AbstractsAminopeptidase and phosphatase activities in basins of Lake Hiidenvesi dominated by cyanobacteria and in laboratory grown AnabaenaFRESHWATER BIOLOGY, Issue 9 2002JAANA VAITOMAA 1.,Extracellular enzyme activities were examined in freshwater basins representing a transition from hypertrophy to mesotrophy and in axenic cyanobacterial cultures to evaluate the ecological role of extracellular enzyme activities of cyanobacteria. 2.,Aminopeptidase activity was related to the trophic status of the lake basins. The activity was highest in the most eutrophic basin and decreased in the less nutrient-rich basins. Cyanobacteria were the most important autotrophic organisms and aminopeptidase activity was positively associated with cyanobacterial biomass. 3.,In an axenic Anabaena batch culture, nitrogenase activity was several orders of magnitude higher than leucine aminopeptidase activity. Nitrate did not have an effect on aminopeptidase activity or growth, but significantly reduced the rate of nitrogen fixation. A high phosphorus concentration at the beginning of the Anabaena batch-culture experiment resulted in reduced phosphatase activity. 4.,In Lake Hiidenvesi, aminopeptidase activity probably originated mostly from attached bacteria and less so from cyanobacteria. [source] Human brain aminopeptidase A: biochemical properties and distribution in brain nucleiJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Nadia De Mota Abstract Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca2+. Kinetic studies showed that the Km (190 ,mol/L) of the human enzyme for the synthetic substrate- l -glutamyl-,-naphthylamide was close from that of the mouse enzyme (256 ,mol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension. [source] Activity-Based Protein Profiling for Type I Methionine Aminopeptidase by Using Photo-Affinity Trimodular ProbesCHEMBIOCHEM, Issue 12 2007Wen-Wei Qiu Dr. Three in one. A trimodular probe (1) with three functional groups (for target recognition, proximal target crosslinking, and distal reporter tag attachment via click chemistry of a photostable azido-acetyl group) has been designed. We demonstrate its specificity, sensitivity, and potential for general application in activity-based protein profiling for type I methionine aminopeptidases. [source] Specific Nonpeptide Inhibitors of Puromycin-Sensitive Aminopeptidase with a 2,4(1H,3H)-Quinazolinedione Skeleton.CHEMINFORM, Issue 15 2004Hiroki Kakuta Abstract For Abstract see ChemInform Abstract in Full Text. [source] Human skeletal muscle cell differentiation is associated with changes in myogenic markers and enhanced insulin-mediated MAPK and PKB phosphorylationACTA PHYSIOLOGICA, Issue 4 2004L. Al-Khalili Abstract Aim:, We hypothesized that myogenic differentiation of HSMC would yield a more insulin responsive phenotype. Methods:, We assessed expression of several proteins involved in insulin action or myogenesis during differentiation of primary human skeletal muscle cultures (HSMC). Results:, Differentiation increased creatine kinase activity and expression of desmin and myocyte enhancer factor (MEF)2C. No change in expression was observed for big mitogen-activated protein kinase (BMK1/ERK5), MEF2A, insulin receptor (IR), hexokinase II, and IR substrates 1 and 2, while expression of glycogen synthase, extracellular signal-regulated kinase 1 and 2 (ERK1/2 MAP kinase) and the insulin responsive aminopeptidase increased after differentiation. In contrast to protein kinase B (PKB)a, expression of (PKB)b increased, with differentiation. Both basal and insulin-stimulated PI 3-kinase activity increased with differentiation. Insulin-mediated phosphorylation of PKB and ERK1/2 MAP kinase increased after differentiation. Conclusion:, Components of the insulin-signalling machinery are expressed in myoblast and myotube HSMC; however, insulin responsiveness to PKB and ERK MAP kinase phosphorylation increases with differentiation. [source] Characterization of alanyl aminopeptidase from insecticide resistant and susceptible strains of Musca domestica L.ENTOMOLOGICAL RESEARCH, Issue 3 2008Sohail AHMED Abstract To investigate the high activity of intracellular proteases in insecticide resistant strains of Musca domestica L., purification by anion-exchange chromatography and gel filtration of one of the enzymes, alanyl aminopeptidase (Ala AP), in three strains of Musca domestica was carried out. The fractions collected by gel filtration of soluble homogenates of the three strains (571ab, 17bb and Cooper) showed a single peak of Ala AP activity. Partially purified Ala AP of the three strains showed high activity at pH 7.5. The presence or absence of Ca2+ in the assay medium did not produce any difference in activity of Ala AP in the 571ab and Cooper strains, but there was a significant difference in the 17bb strain. The activity of Ala AP in all three strains was essentially unaltered in the presence of inhibitors of serine (PMSF), cysteine (E-64) proteases and carboxypeptidases (pepstatin). Ala AP hydrolyzed alanine amino methylcoumarin (Ala-AMC) maximally, followed by phenyl alanine amino methylcoumarin (Phe-AMC), leucyl amino methylcoumarin (Leu-AMC) and ornithine amino methylcoumarin (Orn-AMC). Ala AP from the three strains showed differential activity towards various substrates. The comparison of alanyl aminopeptidase's activity from different sources is discussed. [source] In vivo characterization of the angiotensin-(1,7)-induced dopamine and ,-aminobutyric acid release in the striatum of the ratEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 3 2005Bart Stragier Abstract The effect of angiotensin (Ang)-1,7 on dopamine, ,-aminobutyric acid (GABA) and glutamate release in the striatum of the rat was examined using in vivo microdialysis. Ang-(1,7) was administered locally in the striatum through the microdialysis probe. At a concentration of 100 µm, Ang-(1,7) caused a significant increase in extracellular dopamine and GABA but had no effect on glutamate release. The Ang-(1,7)-induced dopamine release was blocked by EC33, an inhibitor of aminopeptidase A, an enzyme which converts Ang-(1,7) into Ang-(3,7), suggesting that this effect occurs after metabolism into Ang-(3,7). Indeed, administration of Ang-(3,7) (10,100 µm) into the striatum caused a more potent increase in the striatal dopamine release than Ang-(1,7). Because Ang-(3,7) is an inhibitor of insulin-regulated aminopeptidase (IRAP) and because Ang IV, another IRAP inhibitor, also causes a concentration-dependent increase in dopamine in the rat striatum, IRAP may be involved in this effect. In contrast, EC33 had no effect on the Ang-(1,7)-induced GABA increase but the GABA release was blocked by the putative AT1-7 receptor antagonist A779 (0.1 µm) and by the nitric oxide synthase inhibitor L-NAME (1 mm). These drugs could not block the effect of Ang-(1,7) on the striatal dopamine release suggesting that only the observed effects on GABA release are mediated by the AT1-7 receptor and/or are associated with a release of nitric oxide. [source] Identification of yeast aspartyl aminopeptidase gene by purifying and characterizing its product from yeast cellsFEBS JOURNAL, Issue 1 2006Ryo Yokoyama Aspartyl aminopeptidase (EC 3.4.11.21) cleaves only unblocked N-terminal acidic amino-acid residues. To date, it has been found only in mammals. We report here that aspartyl aminopeptidase activity is present in yeast. Yeast aminopeptidase is encoded by an uncharacterized gene in chromosome VIII (YHR113W, Saccharomyces Genome Database). Yeast aspartyl aminopeptidase preferentially cleaved the unblocked N-terminal acidic amino-acid residue of peptides; the optimum pH for this activity was within the neutral range. The metalloproteases inhibitors EDTA and 1.10-phenanthroline both inhibited the activity of the enzyme, whereas bestatin, an inhibitor of most aminopeptidases, did not affect enzyme activity. Gel filtration chromatography revealed that the molecular mass of the native form of yeast aspartyl aminopeptidase is ,,680 000. SDS/PAGE of purified yeast aspartyl aminopeptidase produced a single 56-kDa band, indicating that this enzyme comprises 12 identical subunits. [source] Immunisation with non-integral OMPs promotes pulmonary clearance of Pseudomonas aeruginosaFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2-3 2003Linda D. Thomas Abstract Pseudomonas aeruginosa is an opportunistic bacterial pathogen that can cause fatal acute lung infections in critically ill individuals. Lung damage due to chronic infections in cystic fibrosis sufferers is the major cause of morbidity and mortality in this group. The bacterium produces various immunomodulatory products that enable it to survive in the lung. Innate and increasing resistance to antibiotic therapy shown by this organism heightens the need for development of a vaccine. This study reports the identification of six non-integral protein antigens; Pa 13, azurin, acyl carrier protein (ACP), amidase, aminopeptidase and KatE, purified from a mucoid strain of P. aeruginosa. N-terminal amino acid sequencing was used to identify these proteins and, based on their ascribed functions, determined that their normal cellular location was cytosolic. A rat model of acute pulmonary infection was used to investigate the ability of these protein antigens to enhance pulmonary clearance of a live P. aeruginosa challenge. Mucosal immunisation with four of the six antigens significantly enhanced bacterial clearance from both the lavage fluid and lung tissue. The greatest level of clearance was demonstrated for the antigens; KatE, aminopeptidase and amidase. Enhanced bacterial clearance was maintained when the antigens amidase and aminopeptidase were produced in recombinant form. When delivered parenterally, aminopeptidase demonstrated its continued efficacy as a vaccine candidate. This study has demonstrated that non-integral outer membrane proteins are antigenic and protective and warrant further investigation as potential components of a vaccine. [source] Functional microbial community response to nutrient pulses by artificial groundwater recharge practice in surface soils and subsoilsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2010Kirsten Schütz Abstract Subsurface microorganisms are essential constituents of the soil purification processes associated with groundwater quality. In particular, soil enzyme activity determines the biodegradation of organic compounds passing through the soil profile. Transects from surface soil to a depth of 3.5 m were investigated for microbial and chemical soil characteristics at two groundwater recharge sites and one control site. The functional diversity of the microbial community was analyzed via the activity of eight enzymes. Acid phosphomonoesterase was dominant across sites and depths, followed by l -leucine aminopeptidase and ,-glucosidase. Structural [e.g. phospholipid fatty acid (PLFA) pattern] and functional microbial diversities were linked to each other at the nonwatered site, whereas amendment with nutrients (DOC, NO3,) by flooding uncoupled this relationship. Microbial biomass did not differ between sites, whereas microbial respiration was the highest at the watered sites. Hence, excess nutrients available due to artificial groundwater recharge could not compensate for the limitation by others (e.g. phosphorus as assigned by acid phosphomonoesterase activity). Instead, at a similar microbial biomass, waste respiration via overflow metabolism occurred. In summary, ample supply of carbon by flooding led to a separation of decomposition and microbial growth, which may play an important role in regulating purification processes during groundwater recharge. [source] Community composition and activity of prokaryotes associated to detrital particles in two contrasting lake ecosystemsFEMS MICROBIOLOGY ECOLOGY, Issue 3 2006Charles Lemarchand Abstract The composition, distribution and extracellular enzyme activities of bacteria attached to small (2,50 ,m in size) transparent exopolymer and Coomassie-stained proteinaceous particles (TEP and CSP) were examined in two lakes of different trophic status located in the Massif Central of France. TEP concentrations (104,106 particle per L) were significantly higher in the more productive lake and were significantly related to chlorophyll a concentrations. The majority of TEP and CSP were colonized by bacteria that constituted 2.6% and 7.4% of the total 4,,6-diamidino-2-phenylindole-stained bacteria in lakes Pavin and Aydat, respectively. In both lakes, the composition of particle-associated bacteria was different from that of free-living bacteria, the Betaproteobacteria and Bacteroidetes (i.e. former Cytophaga,Flavobacteria group) being the dominant groups on particles. We also found that 2,5 ,m TEP were more colonized than 2,5 ,m CSP in the two lakes, and that TEP colonization was higher in the less productive lake. Measurements of Leucine aminopeptidase and ,-glucosidase activities in fractionated lake water (0.2,1.2, 1.2,5 and >5 ,m fractions) indicated that proteolytic activity was always higher and that particle-associated bacteria have higher enzymatic activities than free-living bacteria. The glycolytic activities in the 1.2,5 and >5 ,m fractions were related to the abundance of TEP. We conclude that small freshwater detrital organic particles constitute microhabitats with high bacterial activities in pelagic environments and, undoubtedly, present significant ecological implications for the prokaryotic community structure and function in aquatic ecosystems. [source] Exoenzyme activities as indicators of dissolved organic matter composition in the hyporheic zone of a floodplain riverFRESHWATER BIOLOGY, Issue 8 2010SANDRA M. CLINTON Summary 1. We measured the hyporheic microbial exoenzyme activities in a floodplain river to determine whether dissolved organic matter (DOM) bioavailability varied with overlying riparian vegetation patch structure or position along flowpaths. 2. Particulate organic matter (POM), dissolved organic carbon (DOC), dissolved oxygen (DO), electrical conductivity and temperature were sampled from wells in a riparian terrace on the Queets River, Washington, U.S.A. on 25 March, 15 May, 20 July and 09 October 1999. Dissolved nitrate, ammonium and soluble reactive phosphorus were also collected on 20 July and 09 October 1999. Wells were characterised by their associated overlying vegetation: bare cobble/young alder, mid-aged alder (8,20 years) and old alder/old-growth conifer (25 to >100 years). POM was analysed for the ash-free dry mass and the activities of eight exoenzymes (,-glucosidase, ,-glucosidase, , -N-acetylglucosaminidase, xylosidase, phosphatase, leucine aminopeptidase, esterase and endopeptidase) using fluorogenic substrates. 3. Exoenzyme activities in the Queets River hyporheic zone indicated the presence of an active microbial community metabolising a diverse array of organic molecules. Individual exoenzyme activity (mean ± standard error) ranged from 0.507 ± 0.1547 to 22.8 ± 5.69 ,mol MUF (g AFDM),1 h,1, was highly variable among wells and varied seasonally, with the lowest rates occurring in March. Exoenzyme activities were weakly correlated with DO, DOC and inorganic nutrient concentrations. 4. Ratios of leucine aminopeptidase : ,-glucosidase were low in March, May and October and high in July, potentially indicating a switch from polysaccharides to proteins as the dominant component of microbial metabolism. 5. Principal components analysis indicated that there were patch effects and that these effects were strongest in the summer. 6. DOM degradation patterns did not change systematically along hyporheic flowpaths but varied with overlying forest patch type in the Queets River hyporheic zone, suggesting that additional carbon inputs exist. We hypothesise that the most likely input is the downward movement of DOM from overlying riparian soils. Understanding this movement of DOM from soils to subsurface water is essential for understanding both the hyporheic metabolism and the carbon budget of streams and rivers. [source] Characterization of the proteases in the midgut of the xylophagous larvae of Oemona hirta (Coleoptera: Cerambycidae)INSECT SCIENCE, Issue 5 2009Brian David Shaw Abstract, The protein digestive capability of the larvae of the longhorn beetle (Oemona hirta, Coleoptera: Cerambycidae, Fabricius, 1775) was investigated. This species feeds only on wood where there is a high proportion of vascular tissue. The pH of the midgut, the major digestive organ, was alkaline and protein hydrolysis was maximal at alkaline pH. Use of specific synthetic peptide substrates showed that the major protease activities were the endopeptidases, trypsin and chymotrypsin-like activity, and the exopeptidase, leucine aminopeptidase and the pH curves corresponded to that with protein substrate. Studies using a range of serine protease inhibitors as well as specific inhibitors of metalloproteases, cysteine proteases and aspartate proteases confirmed a serine protease-based digestive system similar to earlier reports of sapwood-feeding Cerambycids. Control of these insect pests using protease inhibitors is discussed. [source] Novel inhibitors targeted to methionine aminopeptidase 2 (MetAP2) strongly inhibit the growth of cancers in xenografted nude modelINTERNATIONAL JOURNAL OF CANCER, Issue 1 2005Eunyoung Chun Abstract Inhibition of angiogenesis is emerging as a promising strategy for the treatment of cancer. In our study reported here, the effects of 4 highly potent methionine aminopeptidase 2 (MetAP2) inhibitors, IDR-803, IDR-804, IDR-805 and CKD-732 (designed by structure-based molecular modeling), on angiogenesis and tumor growth were assessed. Concentrations of these inhibitors as low as 2.5 nM were able to inhibit the growth of human umbilical vein endothelial cells (HUVEC) by as much as 50%, arresting growth in the G1 stage of mitosis. An intracellular accumulation of p21WAF1/Cip1 protein was also observed. Furthermore, at higher concentrations (25 nM) of these 4 MetAP2 inhibitors, a significant induction of apoptosis was apparent in the same HUVEC cultures. As a result of these findings, the possible anticancer effects of these inhibitors were examined, utilizing the SNU-398 hepatoma cell line. Interestingly, pretreatment with these inhibitors led to an increased number of apoptotic cells of up to 60% or more, compared to untreated controls. Moreover, utilizing an in vivo xenografted murine model, these inhibitors suppressed the growth of engrafted tumor. In conclusion, these 4 inhibitory compounds potently exert an antiangiogenic effect to inhibit the growth of cancers in vivo and could potentially be useful for the treatment of a variety of cancers. © 2004 Wiley-Liss, Inc. [source] Peptidase activity in various species of dairy thermophilic lactobacilliJOURNAL OF APPLIED MICROBIOLOGY, Issue 2 2004M. Gatti Abstract Aims:, The aim of the present work was to evaluate the enzymatic potential manifested by aminopeptidase activity of different thermophilic Lactobacillus biotypes and to measure the influence of cell growth phase on enzyme expression. Methods and Results:, The activities were evaluated by the hydrolysis of , -naphthylamide substrates for both whole and mechanically disrupted cells of L. helveticus, L. delbrueckii subsp. bulgaricus and L. delbrueckii subsp. lactis strains, collected from both the exponential and the stationary growth phase. In general, activities were higher for cells in the exponential rather than in the stationary phase and the disrupted cells showed higher activities than the whole cells. The highest activity expressed by all strains corresponded to X-prolyl-dipeptidyl aminopeptidase while a moderate activity was observed towards Arg- ,Na, Lys- ,Na and Leu- ,Na. The lowest activity was observed for Pro- ,Na. Conclusions:, It may be inferred that the cell structure and the cell physiology are crucial to define the level of efficiency of expression for aminopeptidase activity. The two species may be characterized by a different enzymatic system that hydrolyses N-terminal leucine. Significance and Impact of the Study:, The differences of peptidase activities in L. helveticus and L. delbrueckii species acquires an importance to comprehend their role in the biochemical events occurring in cheese ripening. [source] Expression of brush border enzymes in response to lead exposure in rat intestineJOURNAL OF APPLIED TOXICOLOGY, Issue 5 2005Priya Kapur Abstract The effect of feeding lead (50 mg kg,1 body weight) daily for 7 days on the development of various brush border enzymes in the intestine has been studied. The activities of brush border sucrase (P < 0.001), lactase (P < 0.001), , -glutamyl transpeptidase (P < 0.05) and leucine aminopeptidase were reduced (P < 0.05), whereas the alkaline phosphatase level was augmented (P < 0.05) in lead fed rats compared with controls. Kinetic studies with sucrase revealed a low Vmax (0.224 in control and 0.160 units mg,1 protein in lead exposed) with no change in Km (12.6,13.5 mm). Western blot analysis for alkaline phosphatase yielded intense staining of enzyme protein in lead fed rats compared with controls, however, the intensity of the antigen signal was reversed for sucrase under these conditions. These findings suggest that ingestion of lead may interfere with the crypt cell differentiation process thus affecting enzyme functions in the rat intestine. Copyright © 2005 John Wiley & Sons, Ltd. [source] Changes in immune and enzyme histochemical phenotypes of cells in the intestinal mucosa of Atlantic salmon, Salmo salar L., with soybean meal-induced enteritisJOURNAL OF FISH DISEASES, Issue 2 2000A M Bakke-McKellep Extracted soybean meal (SBM) in the diet for Atlantic salmon, Salmo salar L., causes an inflammatory response in the distal intestine. The morphological changes of the epithelial cells and a characterization of the inflammatory cell infiltrate of the distal intestinal mucosa were studied using a panel of enzyme and immunohistochemical markers. The salmon (average body weight 927 g) used in the study were fed either a fishmeal-based diet (control diet) or a diet in which 30% of the fishmeal protein was replaced with SBM protein (SBM diet). In salmon fed SBM, there were markedly reduced enzyme reactivities in the distal intestinal epithelial cells, both in the brush border [5,-nucleotidase (5,N), Mg2+-ATPase, alkaline phosphatase (ALP) and leucine aminopeptidase (LAP)] and in the intracellular structures [alkaline and acid phosphatase, non-specific esterase (NSE) and alanine aminopeptidase (AAP)]. There appeared to be an increased presence of cells of monocytic lineage, including macrophages, as well as neutrophilic granulocytes and immunoglobulin (Ig) M in the lamina propria of the SBM-fed fish. The mid intestine showed little response to the diet. The results suggest that toxic/antigenic component(s) of SBM affect the differentiation of the distal intestinal epithelial cells and may help explain the reduced nutrient digestibilities previously reported in salmonids fed extracted SBM. [source] Effect of Yam (Dioscorea alata Compared to Dioscorea japonica) on Gastrointestinal Function and Antioxidant Activity in MiceJOURNAL OF FOOD SCIENCE, Issue 7 2006Cheng-Chin Hsu ABSTRACT:, Effects of Chinese yam (Dioscorea alata) and Japanese yam (Dioscorea japonica) on gastrointestinal functions including intestinal microflora and intestinal enzymes' activities, as well as antioxidant protection against lipopolysaccharide (LPS)-induced oxidative damage, in Balb/cA mice were examined. In part I, mice were fed yam-supplemented diet for 4 or 8 wk, and killed with carbon dioxide. In part II, mice were fed yam-supplemented diet for 4 wk, and followed by intraperitoneal LPS treatment (i.p. 4 mg/kg bodyweight). The intake of Chinese yam and Japanese yam significantly changed intestinal microflora, in which the colony numbers of Bifidobacterium and Lactobacillus were increased and the colony numbers of Clostridium perfringens were decreased (P < 0.05). The intake of both Chinese and Japanese yams also significantly elevated the activity of leucine aminopeptidase and lipase (P < 0.05), and the activities of sucrase and maltase were increased only in 20% yam-treated groups (P < 0.05). The preintake of yam significantly alleviated subsequent LPS-induced oxidative injury by decreasing lipid oxidation level and fibronectin production and elevating superoxide dismutase activity (P < 0.05). Both Chinese and Japanese yams contained dietary fibers, polyphenols, and flavonoids, which may contribute to the observed gastrointestinal function and antioxidant protection. These results suggest that both Chinese yam and Japanese yam were beneficial for intestinal health and oxidation prevention. [source] Human brain aminopeptidase A: biochemical properties and distribution in brain nucleiJOURNAL OF NEUROCHEMISTRY, Issue 1 2008Nadia De Mota Abstract Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca2+. Kinetic studies showed that the Km (190 ,mol/L) of the human enzyme for the synthetic substrate- l -glutamyl-,-naphthylamide was close from that of the mouse enzyme (256 ,mol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension. [source] Involvement of the somatostatin-2 receptor in the anti-convulsant effect of angiotensin IV against pilocarpine-induced limbic seizures in ratsJOURNAL OF NEUROCHEMISTRY, Issue 4 2006Bart Stragier Abstract The anti-convulsant properties of angiotensin IV (Ang IV), an inhibitor of insulin-regulated aminopeptidase (IRAP) and somatostatin-14, a substrate of IRAP, were evaluated in the acute pilocarpine rat seizure model. Simultaneously, the neurochemical changes in the hippocampus were monitored using in vivo microdialysis. Intracerebroventricularly (i.c.v.) administered Ang IV or somatostatin-14 caused a significant increase in the hippocampal extracellular dopamine and serotonin levels and protected rats against pilocarpine-induced seizures. These effects of Ang IV were both blocked by concomitant i.c.v. administration of the somatostatin receptor-2 antagonist cyanamid 154806. These results reveal a possible role for dopamine and serotonin in the anti-convulsant effect of Ang IV and somatostatin-14. Our study suggests that the ability of Ang IV to inhibit pilocarpine-induced convulsions is dependent on somatostatin receptor-2 activation, and is possibly mediated via the inhibition of IRAP resulting in an elevated concentration of somatostatin-14 in the brain. [source] Kinetic studies on aminopeptidase M-mediated degradation of human hemorphin LVV-H7 and its N -terminally truncated productsJOURNAL OF PEPTIDE SCIENCE, Issue 7 2008Harald John Abstract The human hemorphin LVV-H7 belongs to the class of µ-opiod receptor-binding peptides, which also exhibits significant affinity to insulin-regulated aminopeptidase (IRAP) thereby affecting IRAP inhibition. The inhibitory potency towards IRAP is of pharmaceutical interest for the treatment of Alzheimer's disease. Consecutive N -terminal cleavage of the first two amino acid residues of LVV-H7 affects a drastic increase of the binding affinity (V-H7) but ultimately leads to its complete abolition after cleavage of the next amino acid residue (H7). Therefore, we investigated LVV-H7 truncation by aminopeptidase M (AP-M) identified as a LVV-H7 degrading enzyme potentially regulating hemorphin activity towards IRAP in vivo. Using a selective quantitative multi-component capillary zone electrophoretic method (CZE-UV), we analyzed the AP-M-mediated subsequent proteolysis of the hemorphins LVV-H7 (L32 -F41), VV-H7 (V33 -F41), and V-H7 (V34 -F41) in vitro. Incubations were carried out with synthetic hemorphins applied as single substrates or in combination. Maximum velocities (Vmax), catalytic constants (turnover numbers, kcat), and specific enzyme activities (EA) were calculated. L32 cleavage from LVV-H7 happens more than two-times faster (kcat: 140 min,1 ± 9%, EA: 1.0 U/mg ± 9%) than V33 cleavage from VV-H7 (kcat: 61 min,1 ± 10%, EA: 0.43 U/mg ± 10%) or V32 deletion from V-H7 (kcat: 62 min,1 ± 8%, EA: 0.46 U/mg ± 8%). In contrast, we showed that H7 (Y35 -F41) was neither degraded by porcine AP-M nor did it act as an inhibitor for this enzyme. Determined turnover numbers were in the same dimension as those reported for dynorphin degradation. This is the first time that AP-M-mediated truncation of natural underivatized LVV-H7 and its physiological metabolites was analyzed to determine kinetic parameters useful for understanding hemorphin processing and designing hemorphin-derived drug candidates. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT4 receptorJOURNAL OF PEPTIDE SCIENCE, Issue 7 2007Andreas Axén Abstract Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT4 receptor. Displacement of the C -terminal of angiotensin IV with an o -substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (Ki = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a ,-turn in the C -terminal of its bioactive conformation. Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT4 receptor. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] Participation of the secreted dipeptidyl and tripeptidyl aminopeptidases in asaccharolytic growth of Porphyromonas gingivalisJOURNAL OF PERIODONTAL RESEARCH, Issue 3 2009H. Oda Background and Objective:,Porphyromonas gingivalis secretes gingipains, endopeptidases essential for the asaccharolytic growth of this bacterium. P. gingivalis also secretes dipeptidyl aminopeptidases (DPPIV and DPP-7) and a tripeptidyl aminopeptidase (PTP-A), although their role in asaccharolytic growth is unclear. The present study was carried out to elucidate the role of these dipeptidyl/tripeptidyl aminopeptidases on the asaccharolytic growth of P. gingivalis. Material and Methods:, Knockout mutants for the DPPIV (dpp), dpp7 and/or PTP-A genes were constructed. Brain,heart infusion medium supplemented with sterile hemin and menadione (BHIHM) was used as a complex medium, and the minimal medium used was GA, in which the sole energy source was a mixture of immunoglobulin G and bovine serum albumin. Growth of P. gingivalis was monitored by measuring the optical density of the culture. Results:, All knockout mutants for DPPIV, dpp7 and PTP-A grew as well as strain W83 in BHIHM. In GA, growth of single-knockout and double-knockout mutants was similar to that of W83, whereas growth of a triple-knockout mutant (83-47A) was reduced. We purified recombinant DPPIV and recombinant PTP-A from recombinant Escherichia coli overproducers, and purified DPP-7 from the triple-knockout mutant 83-4A. GA supplemented with the three purified dipeptidyl/tripeptidyl aminopeptidases supported the growth of 83-47A. Conclusion:, DPPIV, DPP-7 and PTP-A contribute to the normal growth of P. gingivalis by cleaving substrate peptides into short-chain polypeptides that are efficient energy sources for P. gingivalis. [source] EXTRACELLULAR ENZYMES OF THE MICROCYSTIS AERUGINOSA PCC 7813 STRAIN ARE INHIBITED IN THE PRESENCE OF HYDROQUINONE AND PYROGALLOL, ALLELOCHEMICALS PRODUCED BY AQUATIC PLANTS,JOURNAL OF PHYCOLOGY, Issue 6 2009Dariusz Dziga Several cyanobacterial species have a high potential to dominate in marine environments and freshwater reservoirs, and the ecological and physiological reasons for this phenomenon are not understood comprehensively. In this study, the ability of a Microcystis aeruginosa Kütz. strain to produce free dissolved enzymes was documented. We have observed that this highly toxic strain releases alkaline phosphatase, leucine aminopeptidase, and ,-glucosidase into the ambient environment. Additionally, the inhibitory activity of selected phenols produced by aquatic plants on the activity of these enzymes was analyzed. The investigated compounds, pyrogallol and, to a lesser degree, hydroquinone, decreased the activity of extracellular enzymes produced by M. aeruginosa, with leucine aminopeptidase being the most sensitive to the inhibitors. The noncompetitive character of enzymatic inhibition suggests that the polyphenols produced by aquatic plants are able to influence the activity of different extracellular or membrane-bound enzymes. [source] Pathogenesis of Potato Gangrene Caused by Phoma exigua var. foveata: II.JOURNAL OF PHYTOPATHOLOGY, Issue 7 2004Activities of some Hydrolases, Dehydrogenases Abstract The location of enzyme activity in gangrene-diseased tubers was determined using the nitrocellulose blotting method. The activity of aminopeptidase and esterase was located in tissues adjacent to dry rot caused by Phoma exigua var. foveata and in other apparently healthy tissues. The activity of glucuronidase, succinic and glucose-6-phosphate dehydrogenases (G-6-PDH), however, was confined to tissues adjacent to the rotted tissue. The pathogen produces very active , - and , -glycosidases, so their highest activity occurred in rotten tissue that was filled with fungal mycelium. Results suggest that all these enzymes are involved in alteration of cell metabolism and the destruction of diseased tuber tissue. [source] l -leucine aminopeptidase production by filamentous Aspergillus fungiLETTERS IN APPLIED MICROBIOLOGY, Issue 6 2005K.M. Nampoothiri Abstract Aims:, To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. Methods and Results:, Twenty-eight Aspergillus strains representing 14 species within the genus were screened for l -leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2·61, 2·59 and 1·30 IU ml,1 for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, l -methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11·39, 17·40 and 13·02 IU g,1 dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65,75°C at pH 8·0,9·0 for all three fungi. Metal ions, Co2+ and Fe2+ in 2 mmol l,1 concentration apparently enhanced the relative enzyme activity and heat stability. Conclusions:, Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. Significance and Impact of the Study:, A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate. [source] Studies on the aminopeptidase activities of Porphyromonas gingivalisMOLECULAR ORAL MICROBIOLOGY, Issue 4 2001D. Grenier Porphyromonas gingivalis is an asaccharolytic bacterium that requires nitrogen substrates as carbon and energy sources. The aims of this study were to investigate the aminopeptidase activities of P. gingivalis and to evaluate the effect of aminopeptidase inhibitors on bacterial growth. Only arginine aminopeptidase and dipeptidyl aminopeptidase IV activities were detected. Experimental evidence was obtained suggesting that the Arg-gingipains of P. gingivalis can function as both an endopeptidase and an aminopeptidase. Firstly, the arginine aminopeptidase activity was found to be inhibited by leupeptin, a well-known inhibitor of Arg-gingipain activity. Secondly, a preparation of Arg-gingipain activity could hydrolyze the chromogenic substrate for arginine aminopeptidase. Lastly, a mutant of P. gingivalis constructed via gene disruption by use of suicide plasmids and deficient in both Arg-gingipain A and B was also devoid of arginine aminopeptidase activity. To investigate the key role of aminopeptidase activities in growth of P. gingivalis, aminopeptidase inhibitors were incorporated in the culture medium prior to inoculation. Bestatin and actinonin were the only ones to inhibit growth of P. gingivalis. Their mechanism of growth inhibition appears to be different but does not involve inhibition of the two major aminopeptidase activities (arginine aminopeptidase and dipeptidyl aminopeptidase IV). [source] GPI-anchored aminopeptidase is involved in the acrosome reaction in sperm of the mussel mytilusedulisMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Tatsuru Togo Abstract The sperm of the mussel Mytilus had hydrolytic activities against substrates for aminopeptidase. Acrosome reaction (AR) was suppressed in the presence of aminopeptidase substrate, Phe-4-methylcoumaryl-7-amide (MCA), and an aminopeptidase inhibitor, bestatin. Treatment of sperm with phosphatidylinositol-specific phospholipase C (PI-PLC) released aminopeptidase activity from sperm and suppressed AR. These results suggest that the enzyme is located on the sperm surface via glycosylphosphatidylinositol (GPI)-anchor and is involved in the AR. Immunoblot analysis showed that tyrosine residues of 40, 59, 68, and 72 kDa proteins were phosphorylated during induction of the AR. The 40 kDa protein was also recognized by anti-c-Src antibody by immunoblotting. The tyrosine phosphorylation of these proteins was inhibited when sperm were inseminated in the presence of Phe-MCA, and by PI-PLC treatment. Treatment of sperm with tyrosine kinase activator, 9,10-dimethyl-1,2-benzanthracene, induced AR, and its inhibitor, genistein, suppressed AR. These results suggest that tyrosine phosphorylation of 40, 59, 68, and 72 kDa proteins, induced by the interaction of GPI-anchored aminopeptidase with oocyte surface, triggers AR in Mytilus sperm. Mol. Reprod. Dev. 67: 465,471, 2004. © 2004 Wiley-Liss, Inc. [source] Quaternary benzo[c]phenanthridine alkaloids as inhibitors of aminopeptidase N and dipeptidyl peptidase IVPHYTOTHERAPY RESEARCH, Issue 1 2002Aleksi Abstract Chelerythrine, sanguinarine and an alkaloid extract from Macleaya cordata,sanguiritrin,were found to be inhibitors of aminopeptidase A and dipeptidyl peptidase IV, while fagaronine inhibited dipeptidyl peptidase IV only. At 50,,M, chelerythrine, sanguinarine and sanguiritrin inhibited aminopeptidase N by 82%, 82%, 88%, DPP IV by 38%, 62%, 57%, and fagaronine by 34%, respectively. When bovine serum albumin (500,,g/mL) was added, the inhibition of both proteases by quaternary benzo[c]phenanthridine alkaloids (QBA) (50,,M) was significantly diminished. Strong interaction of chelerythrine and sanguinarine with bovine and human serum albumin was proved by electrophoretic determination of their respective conditional binding constants. Copyright © 2002 John Wiley & Sons, Ltd. [source] Characterisation of Plasmodium invasive organelles; an ookinete microneme proteomePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2009Kalpana Lal Dr. Abstract Secretion of microneme proteins is essential to Plasmodium invasion but the molecular composition of these secretory organelles remains poorly defined. Here, we describe the first Plasmodium microneme proteome. Purification of micronemes by subcellular fractionation from cultured ookinetes was confirmed by enrichment of known micronemal proteins and electron microscopy. Quantitation of electron micrographs showed >14-fold microneme enrichment compared to the intact ookinete, such that micronemes comprised 85% of the identifiable organelles in the fraction. Gel LC-MS/MS of the most abundant protein constituents of the fraction identified three known micronemal proteins chitinase, CTRP, SOAP, together with protein disulphide isomerase (PDI) and HSP70. Highly sensitive MudPIT shotgun proteomics described a total of 345 proteins in the fraction. M1 aminopeptidase and PDI, the former a recognised target of drug development, were both shown to have a micronemal location by IFA. We further identified numerous proteins with established vesicle trafficking and signaling functions consistent with micronemes being part of a regulated secretory pathway. Previously uncharacterised proteins comprise the largest functional group of the microneme proteome and will include secreted proteins important to invasion. [source] | |||||||||||||||||||||||||||||||