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PubMed1999

Comparison of two in vitro activation systems for protoxicant organophosphorous esterase inhibitors.

In order to perform in vitro testing of esterase inhibition caused by organophosphorous (OP) protoxicants, simple, reliable methods are needed to convert protoxicants to their esterase-inhibiting forms. Incubation of parathion or chlorpyrifos with 0.05% bromine solution or uninduced rat liver microsomes (RLM) resulted in production of the corresponding oxygen analogs of these OP compounds and markedly increased esterase inhibition in SH-SY5Y human neuroblastoma cells. Neither activation system affected cell viability or the activity of AChE or NTE in the absence of OP compounds. Although parathion and chlorpyrifos were activated by RLM, bromine activation required fewer steps and produced more esterase inhibition for a given concentration of chlorpyrifos. However, RLM activation of OP protoxicants produced metabolites other than oxygen analogs and may, therefore, be more relevant as a surrogate for OP biotransformation in vivo. This methodology makes the use of intact cells for in vitro testing of esterase inhibition caused by protoxicant organophosphate compounds a viable alternative to in vivo tests.

Animals↗

PubMed1991

Immunogold labeling of perforin and serine esterases in granulated metrial gland cells.

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by creating large pores on the target plasma membrane. Besides perforin, the granules of CTL and NK cells contain a family of serine esterases. Perforin has also been localized in granulated metrial gland (GMG) cells of the murine embryo implantation site by light microscopic immunostaining. Ultrastructural immunogold labeling with antibodies against perforin and a serine esterase (MTSP 1 or granzyme A) shows that GMG cells contain both perforin and serine esterases in the fine granular matrix of their granules. Perforin has been located in all of the granules, whereas gold particles corresponding to serine esterases have been found in most of the granules. Results from the double immunogold technique indicate that perforin and serine esterases colocalize to most of the same granules in GMG cells. This study supports the view that GMG cells are related to cytolytic lymphocytes.

Animals↗

PubMed1980

Esterase electrophoretic pattern relatedness between Shigella species and Escherichia coli.

Esterases of 57 strains of Shigella dysenteriae, Sh. flexneri, Sh. boydii and Sh. sonnei and 26 strains of Escherichia coli, including the Alkalescens Dispar group, were compared by polyacrylamide-agarose gel electrophoresis. Six types of esterase bands differing in their ability to hydrolyse synthetic substrates and in their sensitivity to heat and to di-isofluoropropyl phosphate were defined. Individual activities and sensitivities of these bands and the apparent molecular weight of the major esterase, estimated to be 58000 by polyacrylamide gradient gel electrophoresis, were identical for both Shigella species and E. coli. One esterase with a molecular weight of 104000 was found in some strains of E. coli. Variations in the number and mobility of bands among Shigella strains defined different esterase patterns (zymotypes) which appeared to be distinct for each species.

Electrophoresis, Polyacrylamide Gel↗

PubMed1983

The relationship between conidial germination and esterase activities in Neurospora crassa.

Esterase activity in rapidly germinating Neurospora conidia was several times higher than the esterase activity in conidia which germinate slowly. Starch gel electrophoresis experiments demonstrated the existence of esterase isoenzymes which are specific to the conidia. These isoenzymes completely disappeared during 20 h of conidial germination at 30 degrees C. Electron microscopy showed the successive breakdown of electron-dense compounds in storage bodies during conidial germination. These observations, taken together, indicate that the electron-dense compounds may be hydrolysed by specific esterases to serve as an endogenous energy and material source for germ tube formation. The levels of esterase activity, however, were not always proportional to the time required for conidial germination, indicating the possibility that additional enzyme systems might also be involved in the initial stages of germination.

Acid Phosphatase↗

PubMed1983

Esterase isoenzyme variation in the genus Saprolegnia, with particular reference to the fish-pathogenic S. diclina-parasitica complex.

Esterase isoenzyme patterns determined by slab gel electrophoresis were compared for nearly 60 Saprolegnia isolates, particularly from the S. diclina-parasitica complex. Consistent differences were found between S. diclina and S. parasitica isolates, with the latter being characterized by having between one and five very fast moving esterase bands that were generally absent from the former. A range of asexual (unidentifiable) isolates, taken from the vicinity of fish hatcheries or from fish lesions, were also examined and their esterase isoenzymes shown to be similar to those of S. parasitica, although usually showing fewer bands. The use of esterase isoenzymes for screening potential fish pathogenic isolates of Saprolegnia is briefly compared with results obtained using oogonium morphology or cyst ornamentation, and the relative merits of each method are discussed. It is proposed that, on the basis of their distinctive cyst coat ornamentation (bundles of long 'boathooks') and esterase isoenzymes, fish lesion isolates can be distinguished from saprophytic isolates, and that analysis of isoenzymes should provide a useful method for the screening of potential pathogenic isolates.

Animals↗

PubMed1997

Three Neocallimastix patriciarum esterases associated with the degradation of complex polysaccharides are members of a new family of hydrolases.

Acetylesterase and cinnamoyl ester hydrolase activities were demonstrated in culture supernatant of the anaerobic ruminal fungus Neocallimastix patriciarum. A cDNA expression library from N. patriciarum was screened for esterases using beta-naphthyl acetate and a model cinnamoyl ester compound. cDNA clones representing four different esterase genes (bnaA-D) were isolated. None of the enzymes had cinnamoyl ester hydrolase activity, but two of the enzymes (BnaA and BnaC) had acetylxylan esterase activity, bnaA, bnaB and bnaC encode proteins with several distinct domains. Carboxy-terminal repeats in BnaA and BnaC are homologous to protein-docking domains in other enzymes from Neocallimastix species and another anaerobic fungus, a Piromyces sp. The catalytic domains of BnaB and BnaC are members of a recently described family of Ser/His active site hydrolases [Upton, C. & Buckley, J.T. (1995). Trends Biochem Sci 20, 178-179]. BnaB exhibits 40% amino acid identity to a domain of unknown function in the CelE cellulase from Clostridium thermocellum and BnaC exhibits 52% amino acid identity to a domain of unknown function in the XynB xylanase from Ruminococcus flavefaciens. BnaA, whilst exhibiting less than 10% overall amino acid identity to BnaB or BnaC, or to any other known protein, appears to be a member of the same family of hydrolases, having the three universally conserved amino acid sequence motifs. Several other previously described esterases are also shown to be members of this family, including a rhamnogalacturonan acetylesterase from Aspergillus aculeatus. However, none of the other previously described enzymes with acetylxylan esterase activity are members of this family of hydrolases.

Amino Acid Sequence↗

PubMed1984

Two-dimensional separation of alpha-naphthyl acetate esterases in human leucocytes and platelets.

Normal human leucocytes and platelets contain esterases which hydrolyse alpha-naphthyl acetate (alpha NA). Purified preparations from these cells were investigated by isoelectric focusing and subsequent polyacrylamide gradient gel electrophoresis at pH 9.0. Extractable alpha NA esterases were separated according to isoelectric point (pI) and molecular weight (MW). Monocytes, lymphocytes, granulocytes and platelets contain a unique pattern of alpha NA esterases, most of which can be inhibited by diisopropyl fluorophosphate (DFP; 0.1 mM). Their activity, however, is not affected by eserine (0.1 mM) or p-hydroxymercuribenzoate (1 mM). No protease activity of these enzymes was detected; it is likely that the majority constitute carboxylesterases (EC 3.1.1.1). Monocytes contain five alpha NA esterases which are additionally inhibited by bis(4-nitrophenyl)-phosphate (0.1 mM) and sodium fluoride (40 mM). PIs are in the range 5.7-6.2 and MWs are 145 000, 155 000, 250 000, 290 000 and 340 000. These enzymes are specific for monocytes. Platelets are characterized by a group of alpha NA esterases having pIs between 6.5 and 8.0, these corresponding to MWs ranging from 15 000 to 400 000.

Blood Platelets↗

PubMed1991

Selective action of androgens on the molecular forms of esterases characterized by two-dimensional gel electrophoresis in the epididymis and vas deferens of the mouse.

Molecular forms of esterases were resolved in non-denaturing conditions by using two-dimensional gel electrophoresis with isoelectric focusing in the first dimension and a time-dependent polyacrylamide gradient gel electrophoresis (PAGGE) in the second dimension. This procedure was used to analyse sequential changes in esterase composition along the excurrent genital duct of the mouse and to initiate a specific identification of the androgen-regulated molecular forms. Almost all the 68 variants (pH 3.9-6.4 and 50-300 kDa) revealed by alpha-naphtyl acetate from the fluids of the three parts of the epididymis (caput, corpus, cauda) and vas deferens, could be assigned to the carboxylesterase group as shown by their action on various substrates and sensitivity to inhibitors. Some of these variants co-migrated with those in the serum and testis, whereas other enzyme forms made their first appearance in the caput (13), in the corpus (26) and in the vas deferens (3). The major changes occurred between the caput and the corpus of the epididymis. Only a few acidic spots were not revealed after neuraminidase digestion. Castration of mice (4 weeks) resulted in inhibition of the activity of 34 esterase forms, and thus abolished most of the regional differences in the excurrent duct system. By re-initiating or repressing the synthesis of regional esterase variants, testosterone supplementation (2 and/or 4 weeks) of castrated animals restored the normal esterase pattern in the three epididymal parts, but not in the vas deferens. The major effect of efferent duct ligation (4 weeks) was the emergence in the corpus and cauda of the epididymis of two variants found in the caput of uncastrated mice.

Androgens↗

PubMed1995

Degradation of triglycerides by a pseudomonad isolated from milk: the roles of lipase and esterase studied using recombinant strains over-producing, or specifically deficient in these enzymes.

The roles of lipase and esterase in causing hydrolytic spoilage of milk by a highly lipolytic psychrotrophic strain of Pseudomonas fluorescens, LS107d2, has been studied. Strains of LS107d2 have been constructed that over-produce, or are specifically deficient in, a lipase (encoded by lipA) and an esterase (encoded by estA). Southern blot analysis reveals that LS107d2 contains only one esterase and one lipase (encoded by estA and lipA) and this was confirmed by the phenotypes of mutants on triolein and tributyrin-containing agar. Analysis of broth cultures showed that the lipase is secreted into the culture medium; in contrast, the esterase is not secreted. Free fatty acid (FFA) levels in whole milk cultures of wild-type, over-producing and the mutant strains of LS107d2 have been examined. From these studies it is concluded that esterase is not involved in the accumulation of FFA by hydrolysing short chain fatty acid esters; that the highly lipolytic phenotype of LS107d2 is due solely to a single secreted lipase; and that the main FFA accumulated in milk cultures of LS107d2 are C4, C16, C18 and C18: 1. Evidence is also presented demonstrating that FFA degradation, as well as production, determines the level of FFA in milk contaminated with lipolytic organisms.

Animals↗

PubMed2004

Cloning and expression of a novel esterase gene cpoA from Burkholderia cepacia.

AIMS: To screen and clone a novel enzyme with specific activity for the resolution of (R)-beta-acetylmercaptoisobutyrate (RAM) from (R,S)-beta-acetylmercaptoisobutyrate [(R,S)-ester]. METHODS AND RESULTS: A micro-organism that produces a novel esterase was isolated and identified as the bacterium Burkholderia cepacia by using the analysis of cellular fatty acids, Biolog automated microbial identification/characterization system, and 16S rRNA gene sequence analysis. A novel esterase gene was cloned from the chromosomal DNA of B. cepacia and was designated as cpoA. The cpoA encodes a polypeptide of 273 amino acids which shows a strong sequence homology with many bacterial nonhaeme chloroperoxidases. In addition, a typical serine-hydrolase motif, Gly-X-Ser-X-Gly, and the highly conserved catalytic triad, Ser95, Asp224, and His253, were identified in the deduced amino acid sequence of cpoA by multiple sequence alignment. CONCLUSION: The cpoA cloned from B. cepacia encodes a novel esterase which is highly related to the nonhaeme chloroperoxidases. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that describes the isolation and cloning of a serine esterase gene from B. cepacia, which is useful in the chiral resolution of (R,S)-ester. The cloned gene will allow additional research on the bifunctionality of the enzyme with esterase and chloroperoxidase activity at the structural and functional levels.

Amino Acid Sequence↗

PubMed1976

Release of chymotrypsin-like esterase from isolated rat peritoneal mast cells by arginine-rich calf thymus histone.

Release of chymotrypsin-like esterase from isolated rat peritoneal mast cells by arginine-rich and lysine-rich calf thymus histones was investigated. The arginine-rich histone released the esterase, but the lysine-rich histone did not release the esterase from the mast cells. The isolated mast cells spontaneously released the esterase gradually. The chymotrypsin-like esterase released from the mast cells is considered to digest the destructed material in tissues in a state of chronic destruction, such as chronically inflamed tissues and malignant tumours.

Animals↗

PubMed1976

Purification and properties of an esterase B4 from human liver.

A butyryl esterase, designated B4, has been purified from human liver and some of its properties described. The activity of this enzyme comprises 0.48% of the total butyryl esterase activity found in human liver. Esterase B4 has been distinguished from other butyryl esterases by its preference for the esters of the fluorogenic compounds 4-methyl umbilliferone and fluorescein over naphthyl esters as substrates. Other distinguishing features of this esterase include a relatively high pI (pH 8.7) A monomeric structure of low molecular weight (20 000) and high solubility in solutions of ammonium sulphate.

Amino Acids↗

PubMed1986

Extracellular fluid proteins of goldfish brain: evidence for the presence of proteases and esterases.

Preparations of enriched fractions of extracellular fluid (ECF) proteins from goldfish brain were found to contain protease(s) and esterase(s). The N-substituted furanacryloyl (FA) peptides FA-Phe-Gly-Gly and FA-Phe-OMe were used as model substrates for determining protease and esterase activity, respectively, in a spectrophotometric assay. Studies of the profile of substrate specificity and identification of the types of compounds that were effective as inhibitors showed that these ECF enzymes have some distinctive properties. GSH, but not GSSG, and EDTA inhibited the protease(s) without influencing the esterase(s), whereas L-1-tosylamide-2-phenylethylchloromethyl ketone blocked both protease and esterase activities of ECF. Most of the protease and esterase properties of ECF could be bound to concanavalin A-Sepharose affinity chromatographic columns in association with ependymin--a brain extracellular protein. These observations indicate that ECF may contain a metalloprotease(s) and raise the possibility that the ependymins might be a substrate for these ECF enzymes.

Animals↗

PubMed1972

Activation of arginine and tyrosine esterase in serum from patients with hereditary angio-oedema.

1. The role of clotting factor XII in the activation of the complement subunit C1s to C1 esterase was examined.2. In sera from patients with hereditary angio-oedema who lack the alpha(2)-glycoglobulin C1 inhibitor, silicates and other potent activators of clotting factor XII induced far less C1 esterase activity than did the weaker factor XII activators, carrageenin and cellulose sulphate. In contrast, the intensity of the induced plasma kallikrein activity corresponded more closely to the clot-promoting effect of the factor XII activators.3. Spontaneous generation of C1 esterase activity was only slightly delayed in hereditary angio-oedema sera previously depleted of factor XII. In normal sera, C1 esterase did not develop spontaneously and could not be induced.4. Experiments with inhibitors suggested that the spontaneous activation of C1s may consist of two phases: factor XII and other plasma proteases first activate small amounts of C1s; the resulting C1 esterase then activates the bulk of C1s. The observed spontaneous activation suggests that when fully activated, the C1s present in 1 ml of human serum will hydrolyse 1-2 mumol of ATEe/minute.

Angioedema↗

PubMed1993

Epithelial alpha-naphthyl acetate esterases in the green vervet monkey gingiva before and after periodontal surgery and during tooth eruption.

To provide enzymatic information on de novo formed junctional (JE) and sulcular epithelium (SE), we performed periodontal surgery on 24 teeth. Ten to 14 days postoperatively, all experimental and 16 control teeth were extracted with adjacent buccal gingiva. In addition, specimens from unerupted and partly erupted teeth containing enamel epithelium (EE) were examined. Fixed cryostat sections were cut in series, stained with HE, or incubated with and without substrate for demonstration of alpha-naphthyl acetate esterase activity and for control purposes, respectively. The distribution and intensity of the alpha-naphthyl acetate esterase activity of newly reformed JE and SE was identical to that of the original JE and SE, i.e. suprabasal and very strong. In contrast, both the oral gingival epithelium (OGE) and the EE displayed a very weak enzyme reaction. These observations indicate that the presence of alpha-naphthyl acetate activity of original and reformed JE and SE is probably site specific and of nondevelopmental origin. Heavy inflammation after healing was associated with enhanced epithelial proliferation of OGE and, in addition, marked esterase activity of these proliferations and corresponding OGE. This points at a possible inflammatory induction of the marked esterase activity seen in JE and SE as well as site-specific, connective tissue influences. Further investigation is needed to elucidate the effect of inflammation on the esterase activity.

Animals↗

PubMed1998

Inhibitors of sensillar esterase block reversibly the responses of moth pheromone receptor cells.

The pheromone of Antherae polyphemus is slowly (within min) degraded by a sensillum lymph specific esterase. The effects of two volatile alkyl-thio-trifluoro-methyl-ketones with alkyl chain length of six (HTFP) and ten carbons (DTFP), which selectively inhibit the activity of the sensillar esterase, were tested. Single sensillum recordings from sensilla trichodea were performed on isolated antennae either held in a continuous stream of clean air, or exposed to an airstream containing one of the esterase inhibitors. Afterwards the antennae were stimulated with the pheromone components (E,Z)-6,11-hexadecadienyl acetate and (E,Z)-6,11-hexadecadienal. Both esterase inhibitors caused a reduction of electrophysiological responses to each of the two pheromone compounds. The DTFP was slightly more effective than HTFP. An almost complete block of the receptor potential amplitude and nerve impulse response was achieved within seconds of exposure to the inhibitors. Responses of both pheromone receptor cells were reduced to the same extent. The half-times of rise and decline of the receptor potential remained unaffected in most cells. The responses to pheromone partially recovered to approximately 50-70% within several min in clean air. An inhibition of the sensillar esterase cannot explain the observed effects. The inhibitors could either react directly with the receptor molecules, thereby inhibiting the action of the pheromone, or bind to the pheromone-binding protein (PBP) and prevent the formation of the pheromone-PBP complex stimulating the receptor.

Animals↗

PubMed1995

Characterization of five esterases from Listeria monocytogenes and use of their electrophoretic polymorphism for strain typing.

Esterases from Listeria monocytogenes strains isolated from cheeses were analyzed by starch gel electrophoresis. Five esterases, numbered from EST 1 to EST 5 in order of decreasing anodal migration, were identified. The EST 1, EST 3, EST 4, and EST 5 set was most active toward alpha-naphthyl propionate, while EST 2 was most active toward alpha-naphthyl acetate. Results from inhibitor studies suggest that all of these esterases were EC class 3.1.1.1 carboxylesterases, except that EST 1 and EST 3 also showed some sensitivity to parahydroxymercuribenzoate. Polymorphism of these five esterases was observed in the population. Twelve esterase patterns were defined and used to subdivide serotypes.

Bacterial Typing Techniques↗

PubMed1999

Screening, nucleotide sequence, and biochemical characterization of an esterase from Pseudomonas fluorescens with high activity towards lactones.

A genomic library of Pseudomonas fluorescens DSM 50106 in a lambdaRESIII phage vector was screened in Escherichia coli K-12 for esterase activity by using alpha-naphthyl acetate and Fast Blue RR. A 3.2-kb DNA fragment was subcloned from an esterase-positive clone and completely sequenced. Esterase EstF1 was encoded by a 999-bp open reading frame (ORF) and exhibited significant amino acid sequence identity with members of the serine hydrolase family. The deduced amino acid sequences of two other C-terminal truncated ORFs exhibited homology to a cyclohexanone monooxygenase and an alkane hydroxylase. However, esterase activity was not induced by growing of P. fluorescens DSM 50106 in the presence of several cyclic ketones. The esterase gene was fused to a His tag and expressed in E. coli. The gene product was purified by zinc ion affinity chromatography and characterized. Detergents had to be added for purification, indicating that the enzyme was membrane bound or membrane associated. The optimum pH of the purified enzyme was 7.5, and the optimum temperature was 43 degreesC. The showed highest purified enzyme activities towards lactones. The activity increased from gamma-butyrolactone (18.1 U/mg) to epsilon-caprolactone (21.8 U/mg) to delta-valerolactone (36.5 U/mg). The activities towards the aliphatic esters were significantly lower; the only exception was the activity toward ethyl caprylate, which was the preferred substrate.

Amino Acid Sequence↗