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PubMed2004

Esterase activities in the bivalve mollusc Adamussium colbecki as a biomarker for pollution monitoring in the Antarctic marine environment.

Marine environments are continuously being threatened by a large number of xenobiotics from anthropogenic sources. Even in sparsely populated and relatively pristine areas, such as Antarctica, hazardous chemicals can pose a serious environmental problem. The main aims of the present study were to (1) validate and optimize an analytical technique utilizing a microtitre-plate photometer to ascertain background levels of esterase activities in the Antarctic bivalve Adamussium colbecki, (2) carry out in situ monitoring of esterase activities to assess any potential environmental impacts of the Italian Scientific Antarctic Base "Terra Nova Bay" on the surrounding marine area. Results showed the presence of organophosphorous-sensitive cholinesterase (ChE) and carboxylesterase (CbE) activities in the gills of A. colbecki and optimal assay conditions were comparable with those found for bivalve species from temperate areas. A higher sensitivity of ChE versus acetylthiocholine activity in A. colbecki to chlorpyrifos compared to species from temperate areas may also be inferred. The in situ study indicated no differences in the environmental quality of the three study sites located around the Italian Base.

Animals↗
PubMed2005

Screening and characterization of a novel esterase from a metagenomic library.

Metagenomes from various environmental soils were screened using alpha-naphthyl acetate and Fast Blue RR for a novel ester-hydrolyzing enzyme on Escherichia coli. Stepwise fragmentations and subcloning of the initial insert DNA (30-40 kb) using restriction enzymes selected to exclude already known esterases with subsequent screenings resulted in a positive clone with a 2.5-kb DNA fragment. The cloned sequence included an open reading frame consisting of 1089 bp, designated as est25, encoding a protein of 363 amino acids with a molecular mass of about 38.3 kDa. Amino acid sequence analysis revealed only moderate identity (< or = 48%) to the known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases, such as HGGG (residues 124-127), GxSxG (residues 199-203), and the putative catalytic triad composed of Ser201, Asp303, and His333. Est25 was functionally overexpressed in a soluble form in E. coli with optimal activity at pH 7.0 and 25 degrees C. The purified Est25 exhibited hydrolyzing activity toward p-nitrophenyl (NP)-fatty acyl esters with short-length acyl chains (< or = C6) with the highest activity toward p-NP-acetate (Km=1.0 mM and Vmax = 63.7 U/mg), but not with chain lengths > or = C8, demonstrating that Est25 is an esterase originated most likely from a mesophilic microorganism in soils. Est25 efficiently hydrolyzed (R,S)-ketoprofen ethyl ester with Km of 16.4 mM and Vmax of 59.1 U/mg with slight enantioselectivity toward (R)-ketoprofen ethyl ester. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzymes from a metagenome.

Amino Acid Sequence↗
PubMed2005

A novel esterase from Bacillus subtilis (RRL 1789): purification and characterization of the enzyme.

An esterase (EC 3.1.1.1) produced by an isolated strain of Bacillus subtilis RRL 1789 exhibited moderate to high enantioselectivity in the kinetic resolution of several substrates like aryl carbinols, hydroxy esters, and halo esters. The enzyme named as B. subtilis esterase (BSE), was purified to >95% purity with a specific activity of 944 U/mg protein and 12% overall yield. The purified enzyme is approximately 52 kDa monomer, maximally activity at 37 degrees C and pH 8.0 and fairly stable up to 55 degrees C. The enzyme does not exhibit the phenomenon of interfacial activation with tributyrin and p-nitrophenyl butyrate beyond the saturation concentration. The enzyme showed preference for triacyglycerols and esters of p-nitrophenol with short chain fatty acid. Presence of Ca2+ ions increases the activity of enzyme by approximately 20% but its presence does not have any influence on the thermostability of the enzyme. The enzyme is not a metalloprotein and belongs to the family of serine proteases. The N-terminal amino acid sequence of BSE determined, as Met-Thr-Pro-Glu-Iso-Val-Thr-Thr-Glu-Tyr-Gly- revealed similarity with the N-terminal amino acid sequence of p-nitrobenzylesterase of B. subtilis.

Amino Acid Sequence↗
PubMed2004

Glutathione S-transferases and esterases in placenta after normal and pre-eclamptic pregnancies.

Severe pre-eclampsia reduced significantly (P<0.05) by 68+/-6 per cent (mean+/-sem, n=10) the maximal velocity (V(max)) and, consequently, reduced significantly by 60+/-7 per cent the catalytic efficiency (C(E)) of placental glutathione transferase pi, assayed with ethacrynic acid. Mild and severe pre-eclampsia reduced significantly by 82+/-5 per cent (mean+/-sem, n=5) and by 41+/-5 per cent (mean+/-sem, n=10), respectively, the V(max)and, consequently, reduced significantly by 72+/-7 and by 33+/-13 per cent, respectively, the C(E)of esterase, assayed with p-nitrophenyl acetate. Furthermore, severe pre-eclampsia increased significantly by 296+/-78 per cent the Michaelis-Menten constant (K(m)) of total GST, assayed with chlorodinitrobenzene and, consequently, decreased significantly the C(E)by 83+/-3 per cent. On the other hand, the concentrations of total and non-protein thiols did not change significantly in placental homogenates from patients with mild or severe pre-eclampsia compared to normal pregnancies. These findings would indicate a decreased capacity of the glutathione transferases and esterase detoxification systems to protect the fetus from drugs prescribed to pregnant women suffering pre-eclampsia, mainly in the severe phase.

Adult↗
PubMed2004

Purification of ENOD8 proteins from Medicago sativa root nodules and their characterization as esterases.

ENOD8 proteins were purified from alfalfa (Medicago sativa) root nodules. After extraction of ENOD8 proteins into an aqueous buffer, they were purified by ammonium sulfate precipitation, concanavalin A Sepharose chromatography, and ion-exchange chromatography. Purification was assessed by comparing silver stained SDS-PAGE gels to Western blots developed with a highly specific ENOD8 antibody. Multiple ENOD8 proteins that co-purified were found. ENOD8 proteins were found to have esterase activity, active on acetyl and butyrl esters but not longer chain aliphatic esters. Thus, ENOD8 proteins are unlikely to be lipases. Kinetic analysis showed that ENOD8 proteins esterase activity exhibited Michaelis-Menten kinetics. Considering ENOD8 protein sequence similarity to an exopolygalacturonase/EP4/iEP4 and lanatoside 15'-O-acetylesterase with the results presented here predicts that ENOD8 substrates could be acetylated oligo- or polysaccharides.

Chemical Precipitation↗
PubMed2006

A survey of mutations in the Cochliomyia hominivorax (Diptera: Calliphoridae) esterase E3 gene associated with organophosphate resistance and the molecular identification of mutant alleles.

Cochliomyia hominivorax (Calliphoridae) is one of the most important myiasis-causing flies and is responsible for severe economic losses to the livestock industry throughout the Neotropical region. In Brazil, C. hominivorax has been controlled mainly with organophosphate (OP) insecticides, although the inappropriate use of these chemicals can result in the selection of resistant flies. Changes in carboxylesterase activity have been associated with OP insecticides in some arthopodan species. In this work, we isolated and characterized part of the E3 gene in C. hominivorax (ChalphaE7), which contained the same substitutions responsible for the acquisition of OP hydrolase activity in Lucilia cuprina (Calliphoridae). Digestion of the polymerase chain reaction products with a restriction enzyme that specifically recognized the mutation site unambiguously differentiated wild and mutated esterase alleles. The PCR-RFLP assay therefore provided a fast, reliable DNA-based method for identifying C. hominivorax individuals with a mutation in the esterase gene. Further bioassays to determine the association of this mutation with OP resistance in C. hominivorax should allow the development of more effective strategies for managing this species.

Alleles↗
PubMed1992

Comparison of gram stain, leukocyte esterase activity, and amniotic fluid glucose concentration in predicting amniotic fluid culture results in preterm premature rupture of membranes.

OBJECTIVE: The purpose of this study was to prospectively compare three rapid and inexpensive tests that have been proposed as predictors of amniotic fluid culture results in preterm premature rupture of membranes. STUDY DESIGN: Amniocentesis was performed on 117 patients < or = 34 weeks' gestational age with premature rupture of membranes and no clinical evidence of infection. Amniotic fluid was sent for Gram stain and aerobic, anaerobic, and Mycoplasma cultures. Leukocyte esterase activity and glucose concentration were also determined on the amniotic fluid. RESULTS: Amniotic fluid cultures were positive in 56 patients (47.8%). Leukocyte esterase activity of 1+ or 2+ and an amniotic fluid glucose concentration < or = 16 mg/dl were significantly more sensitive (p < 0.005) than Gram stain in detecting positive amniotic fluid cultures (73%, 68%, and 41%, respectively). CONCLUSIONS: Although each of these rapid tests is useful in assessing for subclinical intraamniotic infection, none of them have sufficient accuracy to make clinical decisions solely on the basis of their results.

Amniotic Fluid↗
PubMed1987

Leukocyte esterase activity in human amniotic fluid for the rapid detection of chorioamnionitis.

Chorioamnionitis plays an important role in perinatal morbidity and mortality. Fast and accurate diagnosis poses a major problem. A prospective study was performed to assess the value of positive leukocyte esterase test (Chemstrip 9) for the diagnosis of chorioamnionitis during labor. We evaluated 21 patients with chorioamnionitis in labor at term and used 21 matched control subjects. The sensitivity and specificity of leukocyte esterase activity were compared with those of amniotic fluid cultures, Gram stains, maternal pyrexia and leukocytosis, and fetal tachycardia. The sensitivity in diagnosing chorioamnionitis was 91% and the specificity was 95%. The use of this test strip could provide a rapid, inexpensive screening test for chorioamnionitis.

Amniotic Fluid↗
PubMed2002

A spectrophotometric assay for feruloyl esterases.

We have developed a spectrophotometric assay for the quantitative determination of feruloyl esterase activity based on release of 4-nitrophenol from a novel substrate, 4-nitrophenyl ferulate in an emulsion of Triton X-100 in aqueous buffer solution. The release of 4-nitrophenol was linear with reaction time at an early stage of the reaction with various esterase preparations. The method proposed here is accurate, rapid, and easy to perform.

Carboxylic Ester Hydrolases↗
PubMed1978

Effects of certain antitumor platinum compounds on kidney esterases.

In a study of the biochemical mechanism of renal toxicity of certain antitumor platinum compounds, particularly cis-dichlorodiammineplatinum(II) (NSC-119875), qualitative and quantitative studies of the soluble nonspecific kidney esterases were carried out using (C57BL/L X DBA/2) mice. There was a major suppression of the testosterone-dependent esterases of treated male mice; these levels dropped to levels below those found in untreated females within 72 h after certain of the drugs were administered. This effect appeared to be in inverse relationship to the numerical value of the LD50 values of the compounds investigated.

Animals↗
PubMed1997

Bile salt activation of human cholesterol esterase does not require protein dimerisation.

Human milk cholesterol esterase (bile salt-activated lipase) plays a role in the dietary uptake of triacylglyceride and cholesteryl ester. The activities toward these substrates are mediated through a unique bile salt-activated mechanism. Previously, it has been proposed that a necessary step in this process is prior protein dimerisation in the presence of primary bile salts. In this study, we addressed the role of protein dimerisation by investigating bile salt interactions on full length and truncated recombinant forms, as analysed by size exclusion chromatography and concanavalin A Sepharose binding experiments. The present findings demonstrate that protein dimerisation is not an obligatory component of the bile salt-activated pathway. A new functional role for the glycosylated C-terminal domain in cholesterol esterase is also demonstrated in the prevention of non-specific hydrophobic interactions.

Bile Acids and Salts↗
PubMed1984

Comparison of low- and high-pressure affinity chromatography for the purification of serine and sulfhydryl esterases.

A series of affinity chromatography packings for the purification of serine and sulfhydryl esterases (acetylcholinesterase, alkaline phosphatase, urokinase and papain) have been synthesized using commercially available agarose, glass and acrylate parent matrices. Two ligands were coupled to the matrices by utilizing carbodiimide or reaction with active groups already present on the matrix: the quaternary ammonium compound trimethyl(p-aminophenyl)ammonium chloride and the serine esterase inhibitor analog p-aminobenzamidine. It was found that enzyme purification on the agarose- or acrylate-based packings was most successful, resulting in as much as fifty-fold purification over starting material. The pressure stability of the acrylate packing allowed purification by high-pressure affinity chromatography and decreased purification times as much as six-fold in comparison to agarose columns.

Acetylcholinesterase↗
PubMed1996

Oleic acid inhibits cholesteryl esterase and cholesterol utilization for testosterone synthesis in mouse Leydig cells.

We have observed that nonesterified fatty acids (NEFA) inhibit testosterone synthesis in response to luteinizing hormone (LH) in mouse Leydig cells, possibly by affecting cholesterol utilization or endogenous concentrations. We have now studied the influence of oleic acid (OA) on the cellular content of cholesterol, hydrolysis of cholesterol esters, and steroidogenesis in isolated mouse Leydig cells. OA (700 micromol/L added with fatty acid-free [FAF] albumin, 3 g/dL) significantly (P<.025) reduced testosterone production in response to LH (10 ng/mL), total cholesterol concentrations of Leydig cells and the culture medium, and cholesteryl esterase activity in the cytosol and mitochondria. We also studied the effects of OA on steroidogenesis and cellular cholesterol concentrations after treatments to increase cellular cholesterol. OA at lower concentrations (5 micromol/L with albumin, 0.1 g/dL) or low-density lipoprotein ([LDL] 4 micrograms protein/mL) increased cellular cholesterol (P<.01) without affecting basal steroidogenesis. These treatments failed to reverse the inhibitory (P<.05) effect of OA on testosterone synthesis following LH stimulation, but did significantly (P<.01) increase cellular cholesterol. In summary, OA appears to inhibit testosterone synthesis by inhibiting cholesteryl esterase activity.

Animals↗
PubMed1990

Histochemical demonstration of non-specific esterases and non-specific acid phosphatases using menadiol substrates.

Although many synthetic substrates and methods are available for the histochemical detection of non-specific esterases and non-specific acid phosphatase, there are still further possibilities to investigate these hydrolases histochemically. This was shown for menadiol diacetate and menadiol diphosphate using tetrazolium salt, simultaneous azo-dye as well as metal salt methods in many rat tissues. In comparison, the azo-dye procedure with various Fast salts or hexazonium Pararosaniline or New Fuchsin delivered less satisfactory results; precisely localized stain in sufficient amounts was obtained for non-specific esterases using nitro BT, tetranitro BT or benzothiazolystyrylphthalhydrazidyl tetrazolium (BSPT) and for non-specific acid phosphatase with BSPT in the tetrazolium salt method or using cerium ions for phosphate trapping in the diaminobenzidine-nickel-hydrogen peroxide procedure.

Acid Phosphatase↗
PubMed1993

Relationship between naphthol AS-D chloroacetate esterase and prostaglandin synthase.

The close topographical correlation between naphthol AS-D chloroacetate esterase-positive macrophages and prostaglandin synthase-rich macrophages in the thymus of normal and cyclosporin-treated rats was observed. It seems possible that chloroacetate esterase is an enzyme related to metabolism of arachidonic acid and/or production of its active metabolites.

Animals↗
PubMed2003

The influence of protein adsorption and surface modifying macromolecules on the hydrolytic degradation of a poly(ether-urethane) by cholesterol esterase.

Previous investigations have demonstrated that the inflammatory cell derived enzyme, cholesterol esterase (CE) could degrade polyurethanes (PUs) by hydrolyzing ester and urethane bonds. Studies that have investigated the development of protective coatings for PUs have reported that the polymer degradation of polyester-urethanes (PESUs) can be reduced with the use of fluorine containing surface modifying macromolecules (SMMs). Since these latter studies were carried out in the presence of relatively pure enzyme, it has not been shown if SMMs would still provide an enhanced inhibitory effect if surfaces were pre-exposed to plasma proteins. This would be more representative of the in vivo scenario since protein adsorption would occur before the appearance of monocyte-derived macrophages which would be a primary source of esterase activities. The current investigation has focused on studying the influence of fibrinogen (Fg) as a simple model of protein adsorption in order to assess the effect of CE in combination with protein on polyether-urethane (PEU) surfaces. The materials were prepared with and without SMMs, and were pre-coated with Fg prior to carrying out biodegradation studies. The pre-adsorption of Fg onto the modified and non-modified surfaces provided a significant delay in the hydrolytic action of CE onto the PEU substrates. However, the effect was gone by 70 days and by the 126th day of incubation, both Fg coated and non-Fg coated groups had the same level of degradation. The difference between Fg coated and non-coated substrates was much smaller for materials containing SMMs. In addition, the pre-adsorption of Fg did not alter the SMMs' ability to provide a more biostable surface over the 4 month incubation period.

Adsorption↗
PubMed2003

Enzyme induced biodegradation of polycarbonate-polyurethanes: dose dependence effect of cholesterol esterase.

The current study has investigated the influence of esterase activity (80-400units/ml) on the biodegradation of polycarbonate-urethanes (PCNUs) by cholesterol esterase (CE), with a particular interest in studying the influence of different hard segment structures and their contribution to sensitizing the polymer towards enzyme catalyzed hydrolysis. Polycarbonate based polyurethanes were synthesized with varying hard segment content as well as hard segment chemistry based on three different diisocyanates, 1,6-hexane diisocyanate (HDI), 4,4'-methylene bisphenyl diisocyanate (MDI) and 4,4-methylene biscyclohexyl diisocyanate (HMDI). The effect of different chemistry on surface contact angle was measured in order to define the relative chemical nature of the surfaces. The enzyme dose response was found to be lower when hard segment content in the polymer was high. There was a very strong dependence on enzyme concentration for polyurethanes with different hard segment chemistry, despite the fact that the nature of the hydrolysable polycarbonate segment remained the same. The PCNU which showed the most dramatic dependence on enzyme concentration was synthesized with HMDI. At low enzyme concentration (80units/ml) this material was the most stable of the polymers while at elevated CE concentration (400units/ml) the polymer underwent a catastrophic breakdown. The findings suggested that protein binding on the surfaces was saturated even though enzyme degradation did not achieve saturation on any of the surfaces. The role of protein binding in modulating the hydrolytic action of the enzymes at different activity levels highlights a need for further study in this area.

Cyanates↗
PubMed1991

Effects of three reputed carboxylesterase inhibitors upon rat serum esterase activity.

Rats have very high endogenous levels of serum carboxylesterase (CAE) compared to primates. This difference accounts for the lower sensitivity of rats to toxic organophosphates, which interact with CAE instead of the more critical acetylcholinesterase. Pretreatment of rats with CAE inhibitors potentiates the effects of organophosphates. In this study, the effects of three putative CAE inhibitors, 2-(o-Cresyl)-4H-1:3:2-benzodioxaphosphorin-2-oxide (CBDP), bis-p-nitrophenyl-phosphate (BNPP), and tetraisopropyl pyrophosphoramide (Iso-OMPA), on the hydrolysis of several commercially available substrates were determined. Respective kinetic constants Km and Vmax were derived and effects of inhibitors compared using saturating amounts of substrate. Data presented here indicate significant differences in substrate affinity (Km), reactivity (Vmax), as well as effects of inhibitors. CBDP inhibits hydrolysis of specific naphthyl and paranitrophenyl esters at relatively low concentrations (1-10 microM). In contrast, significantly higher concentrations (mM) of BNPP and Iso-OMPA were required for inhibition of serum esterase activity. Of the inhibitors tested, Iso-OMPA in general exhibited the smallest inhibitory effect on ester hydrolysis. Although inhibition of hydrolysis of specific paranitrophenyl and naphthyl esters occurred in the presence of similar amounts of CBDP, the degree of inhibition differed significantly (50-75% vs. greater than 90%, respectively). These data suggest that there exists in rat serum, a pool of naphthyl ester esterase activity that is very sensitive ex vivo (greater than 90% inhibition) to CBDP and may be very useful in validating a rodent model for soman toxicity.

Animals↗