Enzymes Leucine Aminopeptidase Research Articles

Cloning and catalytic profile of Hyalomma dromedarii leucine aminopeptidase

Ticks have harmful impacts on both human and animal health and cause considerable economic losses. Leucine aminopeptidase enzymes (LAP) play important roles during tick infestation to liberate vital amino acids necessary for growth. The aim of the current study is to identify, express and characterize the LAP from the hard tick Hyalomma dromedarii and elucidate its biochemical characteristics. We cloned an open reading frame of 1560 bp encoding a protein of 519 amino acids. The LAP full-length was expressed in Escherichia coli BL21 (DE3) and purified. The recombinant enzyme (H.d rLAP- 6×His) had a predicted molecular mass of approximately 55 kDa. Purification and the enzymatic characteristics of H.d rLAP- 6×His were studied. The purified enzyme showed maximum activity at 37 °C and pH 8.0–8.5 using Leu-p-nitroanilide as a substrate. The activity of H.d rLAP- 6×His was sensitive to β-mercaptoethanol, dl-dithiothreitol, 1,10- phenanthroline, bestatin HCl, and EDTA and completely abolished by 0.05 % SDS. In parallel, the enzymatic activity was enhanced by Ni2+, Mn2+ and Mg2+, partially inhibited by Na+, Cu2+, Ca2+ and completely inhibited by Zn2+.

Directing chemiluminescent dioxetanes to mitochondria: A cationic luminophore enables in vitro and in vivo detection of cancer cells upon enzymatic activation

A mitochondrion targeted and leucine aminopeptidase (LAP) activatable 1,2-dioxatane based chemiluminescent probe (MCL) for detection of LAP activity in living cancer cells and tumor bearing mice was reported. MCL displayed a selective and sensitive turn-on response in aqueous solutions upon reacting with the LAP enzyme. In cell culture studies, a selective luminescence intensity increase was observed in cancer cell lines, suggesting that MCL can differentiate between cancer and normal cells and allows detection of varying endogenous LAP concentrations. Using fluorescence imaging with a commercial Mitotracker dye, MCL was also shown to localize mitochondria in cancer cell lines. Furthermore, MCL was used to image tumors in mice models. MCL marks not only the first ever example of a mitochondria targeted chemiluminescent probe, but also the first ever example of an organelle targeted 1,2-dioxetane derivative.

Phytoplankton and Bacterial Communities’ Patterns in a Highly Dynamic Ecosystem (Central Mediterranean Sea)

The Straits of Messina (Southern Italy, Mediterranean Sea) are a very complex area: they connect two basins (Tyrrhenian and Ionian) with different hydrographic features and is characterised by upwelling and mixing phenomena. The aim of the study was to evaluate if and how the physical and chemical water conditions and hydrodynamics influenced the phytoplankton and bacterial patterns and the functioning of this ecosystem. During a late winter survey, size-fractionated phytoplankton (from 0.2 to 200 μm) biomass (chlorophyll a), cell densities and species composition as well as total picoplankton abundances, morphotype composition, and activity levels of the enzymes leucine aminopeptidase, β-glucosidase, and alkaline phosphatase were investigated. The obtained results showed a marked diversification among the water masses identified within the Straits area. The analyses of the phytoplankton diversity indices, particularly those based on phylogenetic relationships between species (indices of taxonomic diversity and distinctness), confirmed our findings. In conclusion, the patterns of phytoplankton and bacterial communities provide a suitable approach to evaluate how microbial communities respond to changing environmental scenarios. This tool could be applied to other temperate Mediterranean ecosystems.

Assembly Transformation Jointly Driven by the LAP Enzyme and GSH Boosting Theranostic Capability for Effective Tumor Therapy

Developing intelligent and morphology-transformable nanomaterials that can spatiotemporally undergo stimulus-responsive size transformation holds great promise for improving the tumor delivery efficiency of drugs in vivo. Here, we report a smart size-transformable theranostic probe Ce6-Leu consisting of a leucine amino peptidase (LAP) and glutathione (GSH) dual-responsive moiety, an 1,2-aminothiol group, and a clinically used photosensitizer Ce6. This probe tends to self-assemble into uniform nanoparticles with an initial size of ∼80 nm in aqueous solution owing to the amphiphilic feature. Surprisingly, taking advantage of the biocompatible CBT-Cys condensation reaction, the large nanoprobes can be transformed into tiny nanoparticles (∼23 nm) under the joint action of LAP and GSH in a tumor microenvironment, endowing them with great tumor accumulation and deep tissue penetration. Concomitantly, this LAP/GSH-driven disassembly and size shrinkage of Ce6-Leu can also activate the fluorescence/magnetic resonance signals and the photodynamic effect for enhanced multimodal imaging-guided photodynamic therapy of human liver HepG2 tumors in vivo. More excitingly, the Mn2+-chelating probe (Ce6-Leu@Mn2+) was demonstrated to have the capability to catalyze endogenous H2O2 to persistently release O2 at the hypoxic tumor site, as a consequence improving the oxygen supply to boost the radiotherapy effect. We thus believe that this LAP/GSH-driven size-transformable nanosystem would offer a novel advanced technology to improve the drug delivery efficiency for achieving precise tumor diagnosis and treatment.

A leucine aminopeptidase activatable photosensitizer for cancer cell selective photodynamic therapy action

Activity based photosensitizers (PS) continue to attract great attention as they enable selective photodynamic therapy action on cancer cells while sparing normal cells even under light irradiation. Sensitivity to specific enzymes that are differentially overexpressed in cancer cells is crucial in the design of activatable PSs. In this direction, we report here, for the first time, a leucine aminopeptidase (LAP) activatable PDT agent (HCL), which is a red-shifted, water soluble and photostable brominated hemicyanine derivative. HCL was activated by endogenous LAP enzyme selectively in A549 (lung) and HCT116 (colon) cancer cells containing high LAP levels and induced effective photocytotoxicity with negligible dark toxicity. Furthermore, the fluorescence of the parent bromo-hemicyanine core was restored upon LAP-based activation in cancer cells. On the other side, no remarkable phototoxicity or fluorescence turn-on was detected in healthy L929 cells. Thus, HCL serves as an effective and tumour associated LAP-sensitive phototheranostic agent. We believe different cancer-associated analytes can be utilized in combination with near-IR absorbing scaffolds in the scope of activatable PDT designs to enrich the tumour-selective PS arsenal.

Use of biochar with immobilized free-living N-fixers as mineral fertilizer

It is known that biochar application as soil fertilizer lead to reduce N loss. At the same time because of its porosity biochar is high quality substrate for immobilization microorganisms. The practice of using free-living N fixing bacteria (FLNFB) in organic farming is widely used to increase the nitrogen content in the soil. In this study immobilisation FLNFB on two types of biochar (made of chicken manure and sewage sludge) was applied to increase soil quality and crop productivity. The effectiveness of immobilization has been proven using SEM method: biofilms of FLNFB on the porous surface of both types of biochars are visible in the SEM images. The effectiveness of application 1% by mass biochar with FLNFB was estimated during 30 days vegetation experiment in greenhouse conditions by estimating the chemical composition of the soil (pH, CEC, OM, Corg, Ntot), by estimating change the functional activity of the soil microbial community (soil respiration activity, leucine aminopeptidase enzyme activity), by estimating change the structure of the microbial community (qPCR were used to analyze nifH and 16S rRNA genes in order to study the total count of bacterial and fungi), by estimating change the plants characteristics (chlorophyll content, germination, root and shoot length, biomass). It was found that application biochar with FLNFB lead to higher content of Ntot (0.11-0.19%) in soil in the end of the experiment as comparing than that for soil samples with mineral fertilizer ( 0.09%) and control soil (0.08%). The application biochar with FLNFB lead to significantly increase nifH-genes in soil. The soil leucine aminopeptidase and respiration activity have a similar trend for samples with and without biochar. The use of biochar as fertilizer led to increase chlorophyll content (13-22%) and barley biomass (10-84%) and decrease the length of the root (3-33%) in comparison with the control soil.

Evaluation of Arthrospira sp. enzyme hydrolysate as dietary additive in gilthead seabream (Sparus aurata) juveniles

The aim of this work was to evaluate the effects of the dietary inclusion of Arthrospira sp. enzyme hydrolysate on gilthead seabream (Sparus aurata) juveniles in a 128-day feeding trial. Algal hydrolysate was tested at low inclusion level, namely, 2 and 4%, against a control diet without Arthrospira sp. supplementation. At the end of the feeding trial, fish body weight was recorded for growth evaluation. No significant differences were found among the experimental groups regarding growth performance or nutrient utilization, despite the fact that those animals fed with diets enriched with Arthrospira displayed higher trypsin, chymotrypsin, and leucine aminopeptidase enzyme activities, compared to fish fed with control diet. The ultrastructural study of the intestinal mucosa also revealed increased microvilli length and absorptive capacity in fish fed with Arthrospira sp. diets, especially at 4% inclusion level. Arthrospira supplementation was also responsible for lower lipid oxidation in muscle tissue, as well as for remarkable colour differences in skin, compared to control animals. These results suggest, overall, that low dietary supplementation with this microalgal hydrolysate might improve not only the intestinal ultrastructure and functionality but also muscle pigmentation and antioxidant capacity of juvenile gilthead seabream.

Do elevated CO2 and temperature affect organic nitrogen fractions and enzyme activities in soil under rice crop?

A study was conducted to quantify the effect of elevated carbon dioxide (CO2) and temperature on soil organic nitrogen (N) fractions and enzyme activities in rice rhizosphere. Rice crop was grown inside the open top chambers in the ICAR-Indian Agricultural Research Institute. The N was applied in four different doses. Grain yield and aboveground N uptake by rice significantly reduced under elevated temperature. However, elevated CO2 along with elevated temperature was able to compensate this loss. Principal component analysis clearly indicated that microbial biomass carbon, microbial biomass N, amino acid N, total hydrolysable N, ammonia N and serine–threonine N contributed significantly to rice grain yield. Combined effect of elevated CO2 and elevated temperature decreased the total hydrolysable N, especially for lower N doses. The N-acetyl-glucosaminidase and leucine aminopeptidase enzyme activities were negatively correlated with the organic N pools. Higher activities of these enzymes under limited N supply may accelerate the decomposition of organic N in soil. When N was applied in super-optimal dose, plant N demand was met thereby causing lesser depletion of total hydrolysable N. Better nitrogen management will alleviate faster depletion of native soil N under future scenario of climate change and thus might cause N sequestration in soil.

Design, synthesis, and biological evaluation of water-soluble amino acid prodrug conjugates derived from combretastatin, dihydronaphthalene, and benzosuberene-based parent vascular disrupting agents

Targeting tumor vasculature represents an intriguing therapeutic strategy in the treatment of cancer. In an effort to discover new vascular disrupting agents with improved water solubility and potentially greater bioavailability, various amino acid prodrug conjugates (AAPCs) of potent amino combretastatin, amino dihydronaphthalene, and amino benzosuberene analogs were synthesized along with their corresponding water-soluble hydrochloride salts. These compounds were evaluated for their ability to inhibit tubulin polymerization and for their cytotoxicity against selected human cancer cell lines. The amino-based parent anticancer agents 7, 8, 32 (also referred to as KGP05) and 33 (also referred to as KGP156) demonstrated potent cytotoxicity (GI50=0.11–40nM) across all evaluated cell lines, and they were strong inhibitors of tubulin polymerization (IC50=0.62–1.5μM). The various prodrug conjugates and their corresponding salts were investigated for cleavage by the enzyme leucine aminopeptidase (LAP). Four of the glycine water-soluble AAPCs (16, 18, 44 and 45) showed quantitative cleavage by LAP, resulting in the release of the highly cytotoxic parent drug, whereas partial cleavage (<10–90%) was observed for other prodrugs (15, 17, 24, 38 and 39). Eight of the nineteen AAPCs (13–16, 42–45) showed significant cytotoxicity against selected human cancer cell lines. The previously reported CA1-diamine analog and its corresponding hydrochloride salt (8 and 10, respectively) caused extensive disruption (at a concentration of 1.0μM) of human umbilical vein endothelial cells growing in a two-dimensional tubular network on matrigel. In addition, compound 10 exhibited pronounced reduction in bioluminescence (greater than 95% compared to saline control) in a tumor bearing (MDA-MB-231-luc) SCID mouse model 2h post treatment (80mg/kg), with similar results observed upon treatment (15mg/kg) with the glycine amino-dihydronaphthalene AAPC (compound 44). Collectively, these results support the further pre-clinical development of the most active members of this structurally diverse collection of water-soluble prodrugs as promising anticancer agents functioning through a mechanism involving vascular disruption.

Responses of absolute and specific soil enzyme activities to long term additions of organic and mineral fertilizer

Long-term phosphorus (P) and nitrogen (N) applications may seriously affect soil microbial activity. A long-term field fertilizer application trial was established on reddish paddy soils in the subtropical region of southern China in 1998. We assessed the effects of swine manure and seven different rates or ratios of NPK fertilizer treatments on (1) the absolute and specific enzyme activities per unit of soil organic carbon (SOC) or microbial biomass carbon (MBC) involved in C, N, and P transformations and (2) their relationships with soil environmental factors and soil microbial community structures. The results showed that manure applications led to increases in the absolute and specific activities of soil β-1,4-glucosidase(βG), β-1,4-N-acetylglucosaminidase (NAG), and leucine aminopeptidase (LAP). The absolute and specific acid phosphatase (AP) activities decreased as mineral P fertilizer application rates and ratios increased. Redundancy analysis (RDA) showed that there were negative correlations between absolute and specific AP activities, pH, and total P contents, while there were positive correlations between soil absolute and specific βG, NAG, and LAP enzyme activities, and SOC and total N contents. RDA showed that the contents of actinomycete and Gram-positive bacterium PLFA biomarkers are more closely related to the absolute and specific enzyme activities than the other PLFA biomarkers (P<0.01). Our results suggest that both the absolute and specific enzyme activities could be used as sensitive soil quality indicators that provide useful linkages with the microbial community structures and environmental factors. To maintain microbial activity and to minimize environmental impacts, P should be applied as a combination of inorganic and organic forms, and total P fertilizer application rates to subtropical paddy soils should not exceed 44kgPha−1year−1.

Effects of DL-methionine-supplemented dietary soy protein concentrate on growth performance and intestinal enzyme activity of gilthead sea bream (Sparus aurata L.)

The present study evaluated the effects of partial fish meal (FM) replacement by a soy protein concentrate (SPC) on growth performance and digestive enzyme activity of gilthead sea bream. Isonitrogenous (45 % crude protein) and isoenergetic (23 kJ g−1 gross energy) diets with 20 % (SPC20), 40 % (SPC40) and 60 % (SPC60) of SPC inclusion were evaluated against a diet containing FM as the sole protein source. Diets were allocated in triplicate groups of 26 g fish (8 kg/m3/tank) and administered for 3 months. Growth, feed utilization, carcass proximate composition, serum cholesterol levels and digestive enzyme activities including -amylase, maltase, leucine aminopeptidase and alkaline phosphatase were evaluated. At the end of the trial, final weight (74.5 g) and feed efficiency (FE, 0.78) indices were significantly lower in the SPC60 group compared with the FM (93.2 g and 0.95, respectively). Similarly, body lipid content (9.3 %) and serum cholesterol levels (119 mg/dl) were significantly lower in fish fed the SPC60 diet compared with the FM group (12.3 % and 243 mg/dl, respectively). Concerning enzyme activities, maltase and leucine aminopeptidase were significantly decreased in the pyloric caeca and the anterior section of the intestine in fish from the SPC60 group, while -amylase activity in the anterior and posterior sections (P < 0.05) was significantly increased in the SPC40 and SPC60 groups. Overall, increased dietary inclusion of SPC in gilthead sea bream resulted in a gradual reduction in growth performance, but substantial reductions were not observed until the replacement of FM by SPC exceeded 40 %.

Stimulated bacterial growth under elevated p CO₂: results from an off-shore mesocosm study.

Marine bacteria are the main consumers of freshly produced organic matter. Many enzymatic processes involved in the bacterial digestion of organic compounds were shown to be pH sensitive in previous studies. Due to the continuous rise in atmospheric CO2 concentration, seawater pH is presently decreasing at a rate unprecedented during the last 300 million years but the consequences for microbial physiology, organic matter cycling and marine biogeochemistry are still unresolved. We studied the effects of elevated seawater pCO2 on a natural plankton community during a large-scale mesocosm study in a Norwegian fjord. Nine Kiel Off-Shore Mesocosms for Future Ocean Simulations (KOSMOS) were adjusted to different pCO2 levels ranging initially from ca. 280 to 3000 µatm and sampled every second day for 34 days. The first phytoplankton bloom developed around day 5. On day 14, inorganic nutrients were added to the enclosed, nutrient-poor waters to stimulate a second phytoplankton bloom, which occurred around day 20. Our results indicate that marine bacteria benefit directly and indirectly from decreasing seawater pH. During the first phytoplankton bloom, 5–10% more transparent exopolymer particles were formed in the high pCO2 mesocosms. Simultaneously, the efficiency of the protein-degrading enzyme leucine aminopeptidase increased with decreasing pH resulting in up to three times higher values in the highest pCO2/lowest pH mesocosm compared to the controls. In general, total and cell-specific aminopeptidase activities were elevated under low pH conditions. The combination of enhanced enzymatic hydrolysis of organic matter and increased availability of gel particles as substrate supported up to 28% higher bacterial abundance in the high pCO2 treatments. We conclude that ocean acidification has the potential to stimulate the bacterial community and facilitate the microbial recycling of freshly produced organic matter, thus strengthening the role of the microbial loop in the surface ocean.

Histochemical distribution of intestinal enzymes of juvenile pacu (Piaractus mesopotamicus) fed lyophilized bovine colostrum.

Enzyme activity was evaluated in the intestine of juvenile pacu, Piaractus mesopotamicus, fed diets containing 0, 10 or 20 % of lyophilized bovine colostrum (LBC) inclusion for either 30 or 60 days. The enzymes intestinal acid and alkaline phosphatase (ACP and ALP, respectively), nonspecific esterase (NSE), lipase (LIP), dipeptidyl aminopeptidase IV (DAP IV) and leucine aminopeptidase (LAP) were studied using histochemistry in four intestinal segments (S1, S2, S3 and rectum). Moderate activity of the DAP IV was detected in the three last intestinal segments, but no differences among the treatments were detected. Enzymes LAP, NSE and LIP were weakly stained in all intestinal segments and the inclusion of 10 or 20 % of LBC in the diet commanded a moderate reaction to NSE in the S3 segment at day 60. ACP activity was detected only in the brush border of the S1 segment of fish fed 0 % LBC for either 30 or 60 days. The activity of ALP was very strong in the first intestinal segment, but a weak reaction was seen in the last segments. The inclusion of 20 % of LBC changed the pattern of staining to the ALP, eliciting moderate staining in S2 at day 30 and S1 at day 60. The consumption of diets containing LBC by juvenile pacu did not have significant implications in intestinal enzymatic activity, which still was not fully stimulated.

Alterations in digestive enzyme activities during the development of diet-induced enteritis in Atlantic salmon, Salmo salar L.

This study followed the time-related changes in intestinal digestive physiology of post-smolt Atlantic salmon (Salmo salar L.; initial weight 500–600g) during the development of soybean meal induced enteritis. Intestinal tissue and content (chyme) were sampled after 1, 2, 3, 5, 7, 10, 14, 17, and 21days of dietary exposure to a 20% extracted soybean meal-containing (SBM) diet. Fish fed a fishmeal-based control diet sampled on day 0 (prior to SBM feeding) served as controls. From the different intestinal regions, histomorphological evaluation was conducted, activities of the pancreatic enzymes trypsin, chymotrypsin, elastase and lipase, as well as leucine aminopeptidase (LAP) were determined in chyme, and the brush border membrane (BBM) enzymes LAP and maltase were measured in homogenates of the intestinal wall. Epithelial cell functional differentiation was visually assessed using immunohistochemical expression of carbonic anhydrase 12 (CA-12). Histological signs of SBM-induced inflammation were observed in the distal intestine (DI) from day 5. At each given sampling time, pancreatic enzyme activities generally decreased aborally along the intestinal tract. Within each intestinal region, pancreatic protease activities tended to increase with exposure time to the SBM diet, with the greatest activity increases observed in the DI. The magnitude of the decrease in activity between the most proximal region and the most distal decreased with SBM exposure. DI tissue weight and BBM maltase and LAP activities were significantly reduced from day 1–2 and continued to decrease progressively with increasing SBM exposure time. Immunohistochemical staining of CA-12 also diminished over time starting at day 5. By day 7, very little CA-12 staining was observed. The data suggests an immediate and generalized physiological response to a change in diet and/or feed intake in all regions of the intestine, as well as a more localized functional loss in the DI that progressed with the development of SBM-induced inflammation from day 5. The diet change appeared to cause a rapid pancreatic response with increased secretion of all the enzymes investigated, while the longer term responses of the pancreatic proteases differed from that of lipase.

Low Concentrations of Silver Nanoparticles in Biosolids Cause Adverse Ecosystem Responses under Realistic Field Scenario

A large fraction of engineered nanomaterials in consumer and commercial products will reach natural ecosystems. To date, research on the biological impacts of environmental nanomaterial exposures has largely focused on high-concentration exposures in mechanistic lab studies with single strains of model organisms. These results are difficult to extrapolate to ecosystems, where exposures will likely be at low-concentrations and which are inhabited by a diversity of organisms. Here we show adverse responses of plants and microorganisms in a replicated long-term terrestrial mesocosm field experiment following a single low dose of silver nanoparticles (0.14 mg Ag kg−1 soil) applied via a likely route of exposure, sewage biosolid application. While total aboveground plant biomass did not differ between treatments receiving biosolids, one plant species, Microstegium vimeneum, had 32 % less biomass in the Slurry+AgNP treatment relative to the Slurry only treatment. Microorganisms were also affected by AgNP treatment, which gave a significantly different community composition of bacteria in the Slurry+AgNPs as opposed to the Slurry treatment one day after addition as analyzed by T-RFLP analysis of 16S-rRNA genes. After eight days, N2O flux was 4.5 fold higher in the Slurry+AgNPs treatment than the Slurry treatment. After fifty days, community composition and N2O flux of the Slurry+AgNPs treatment converged with the Slurry. However, the soil microbial extracellular enzymes leucine amino peptidase and phosphatase had 52 and 27% lower activities, respectively, while microbial biomass was 35% lower than the Slurry. We also show that the magnitude of these responses was in all cases as large as or larger than the positive control, AgNO3, added at 4-fold the Ag concentration of the silver nanoparticles.

Response of &amp;lt;i&amp;gt;Nodularia spumigena&amp;lt;/i&amp;gt; to &amp;lt;i&amp;gt;p&amp;lt;/i&amp;gt;CO&amp;lt;sub&amp;gt;2&amp;lt;/sub&amp;gt; – Part 2: Exudation and extracellular enzyme activities

Abstract. The filamentous and diazotrophic cyanobacterium Nodularia spumigena plays a major role in the productivity of the Baltic Sea as it forms extensive blooms regularly. Under phosphorus limiting conditions Nodularia spumigena have a high enzyme affinity for dissolved organic phosphorus (DOP) by production and release of alkaline phosphatase. Additionally, they are able to degrade proteinaceous compounds by expressing the extracellular enzyme leucine aminopeptidase. As atmospheric CO2 concentrations are increasing, we expect marine phytoplankton to experience changes in several environmental parameters, including pH, temperature, and nutrient availability. The aim of this study was to investigate the combined effect of CO2-induced changes in seawater carbonate chemistry and of phosphate deficiency on the exudation of organic matter, and its subsequent recycling by extracellular enzymes in a Nodularia spumigena culture. Batch cultures of Nodularia spumigena were grown for 15 days under aeration with low (180 μatm), medium (380 μatm), and high (780 μatm) CO2 concentrations. Obtained pCO2 levels in the treatments were on median 315, 353, and 548 μatm CO2, respectively. Extracellular enzyme activities as well as changes in organic and inorganic compound concentrations were monitored. CO2 treatment–related effects were identified for cyanobacterial growth, which in turn influenced the concentration of mucinous substances and the recycling of organic matter by extracellular enzymes. Biomass production was increased by 56.5% and 90.7% in the medium and high pCO2 treatment, respectively, compared to the low pCO2 treatment. In total, significantly more mucinous substances accumulated in the high pCO2 treatment, reaching 363 μg Xeq L−1 compared to 269 μg Xeq L−1 in the low pCO2 treatment. However, cell-specific rates did not change. After phosphate depletion, the acquisition of P from DOP by alkaline phosphatase was significantly enhanced. Alkaline phosphatase activities were increased by factor 1.64 and 2.25, respectively, in the medium and high compared to the low pCO2 treatment. We hypothesise from our results that Nodularia spumigena can grow faster under elevated pCO2 by enhancing the recycling of organic matter to acquire nutrients.

Mechanism of peptide hydrolysis by co-catalytic metal centers containing leucine aminopeptidase enzyme: a DFT approach

In this density functional theory study, reaction mechanisms of a co-catalytic binuclear metal center (Zn1-Zn2) containing enzyme leucine aminopeptidase for two different metal bridging nucleophiles (H(2)O and -OH) have been investigated. In addition, the effects of the substrate (L-leucine-p-nitroanilide → L-leucyl-p-anisidine) and metal (Zn1 → Mg and Zn2 → Co, i.e., Mg1-Zn2 and Mg1-Co2 variants) substitutions on the energetics of the mechanism have been investigated. The general acid/base mechanism utilizing a bicarbonate ion followed by this enzyme is divided into two steps: (1) the formation of the gem-diolate intermediate, and (2) the cleavage of the peptide bond. With the computed barrier of 17.8 kcal/mol, the mechanism utilizing a hydroxyl nucleophile was found to be in excellent agreement with the experimentally measured barrier of 18.7 kcal/mol. The rate-limiting step for reaction with L-leucine-p-nitroanilide is the cleavage of the peptide bond with a barrier of 17.8 kcal/mol. However, for L-leucyl-p-anisidine all steps of the mechanism were found to occur with similar barriers (18.0-19.0 kcal/mol). For the metallovariants, cleavage of the peptide bond occurs in the rate-limiting step with barriers of 17.8, 18.0, and 24.2 kcal/mol for the Zn1-Zn2, Mg1-Zn2, and Mg1-Co2 enzymes, respectively. The nature of the metal ion was found to affect only the creation of the gem-diolate intermediate, and after that all three enzymes follow essentially the same energetics. The results reported in this study have elucidated specific roles of both metal centers, the nucleophile, indirect ligands, and substrates in the catalytic functioning of this important class of binuclear metallopeptidases.

Development of digestive enzymes in larvae of Mayan cichlid Cichlasoma urophthalmus

The development of digestive enzymes during the early ontogeny of the Mayan cichlid (Cichlasoma urophthalmus) was studied using biochemical and electrophoretic techniques. From yolk absorption (6days after hatching: dah), larvae were fed Artemia nauplii until 15dah, afterward they were fed with commercial microparticulated trout food (45% protein and 16% lipids) from 16 to 60dah. Several samples were collected including yolk-sac larvae (considered as day 1 after hatching) and specimens up to 60dah. Most digestive enzymes were present from yolk absorption (5-6dah), except for the specific acid proteases activity (pepsin-like), which increase rapidly from 8dah up to 20dah. Three alkaline proteases isoforms (24.0, 24.8, 84.5kDa) were detected at 8dah using SDS-PAGE zymogram, corresponding to trypsin, chymotrypsin and probably leucine aminopeptidase enzymes, and only one isoform was detected (relative electromobility, Rf=0.54) for acid proteases (pepsin-like) from 3dah onwards using PAGE zymogram. We concluded that C. urophthamus is a precocious fish with a great capacity to digest all kinds of food items, including artificial diets provided from 13dah.

Effects of protein hydrolysate in weaning diets for Atlantic cod (Gadus morhuaL.) and Atlantic halibut (Hippoglossus hippoglossusL.)

The study aims to test whether predigested dietary protein enhances the utilization of formulated diets at weaning, and also whether it stimulates intestinal maturation. In this study, Atlantic cod [Gadus morhua L.; 41 days posthatch (dph)] and Atlantic halibut [Hippoglossus hippoglossus L.; 63 days postfirst feeding (dpff)] were weaned onto diets with graded levels of protein hydrolysate. By increasing the inclusion of dietary protein as hydrolysate from 0 to 400 g kg−1, cod increased the rate of survival from 7 ± 1% to 18 ± 2% (82 dph; regression, P = 4*10−7). In halibut, the survival rate decreased from 57 ± 9% to 22 ± 7% as the inclusion of protein in the form of hydrolysate increased from 0 to 450 g kg−1 (119 dpff; regression, P = 8*10−5). Growth was not affected in any of the species. Results in specific activities of the intestinal enzymes leucine aminopeptidase (LAP) and alkaline phospatase (AP) supported the results in survival in halibut and partly also in cod, by showing increased activities in groups with increased survival (anova, P < 0.05). The lower optimal level of hydrolysed protein in halibut than in cod is suggested mainly because of a slower feeding practice in halibut, which allows more extensive nutrient leaching before ingestion.

Leucine Aminopeptidase Is an Immunodominant Antigen of Fasciola hepatica Excretory and Secretory Products in Human Infections

The liver fluke Fasciola hepatica parasitizes humans and ruminant livestock worldwide, and it is now being considered a reemerging zoonotic disease, especially in areas in which it is endemic, such as South America. This study investigates the immune response to excretory and secretory products produced by F. hepatica in a group of patients from the Peruvian Altiplano, where the disease is highly endemic. Using a proteomic approach and immunoblotting techniques, we have identified the enzymes leucine aminopeptidase (LAP) and phosphoenolpyruvate carboxykinase as immunodominant antigens recognized by sera from fasciolosis patients. An indirect enzyme-linked immunosorbent assay using recombinant LAP as the antigen was developed to check sera from individuals of this region. Our results demonstrate that LAP produces a specific and strong reaction, suggesting its potential use in the serologic diagnosis of F. hepatica infections in humans.