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Chemiluminescent determination of esterases in monocytes.

Esterase from monocytes promotes the hydrolysis of 2-methyl-1-propenylbenzoate (MPB) yielding 2-methyl-1-propenol, which is oxidized by horseradish peroxidase/H2O2 producing triplet acetone. The chemiluminescence of this reaction can be enhanced by the addition of 9,10-dibromoanthracene-2-sulphonate. The non-specific esterase present in monocytes is responsible for MPB hydrolysis, since (a) the chemiluminescence of the reaction was inhibited by fluoride, and (b) cells that do not contain a significant amount of non-specific esterases, e.g. lymphocytes and neutrophils, did not trigger light emission. The analytical application of this reaction is considered.

Benzoates↗

Analysis of acid esterase activity and polymorphism as an aid for the classification of human low-grade malignant non-Hodgkin's lymphomas.

Investigating a link between enzyme histochemical and recent immunohistochemical results, the authors studied the activity and the polymorphism of acid esterase (EC 3.1.1.6) in well defined human B-cell lymphomas. TWelve cases of chronic B-lymphocytic leukemia, 18 cases of centroblastic/centrocytic follicular lymphoma, and 17 cases of lymphoplasmacytic/lymphoplasmacytoid lymphoma, as diagnosed according to Kiel classification, were subjected to enzyme assay and isoelectric focusing of acid esterase. Enzyme values revealed no characteristic distribution among the lymphoma entities. The isoenzyme pattern, specific to normal human B-lymphocytes, were regularly detectable in all lymphoma entities. The results document the B-cell origin of the analyzed subsets of B-cell malignancies, although distinctive acid esterase patterns were lacking. The prevalence of three anodal isoenzymes in cases of chronic B-lymphocytic leukemia, hardly detectable in other lymphoma entities, were interpreted as the expression of a clonal proliferation, arrested at a certain differentiation stage of B-cells common for the majority of the tumor cells. Hence, further evidence is provided supporting the view that the studied entities represent B-cell neoplasias at different stages of differentiation expressing a variety of different markers.

B-Lymphocytes↗

Occurrence of particular isoenzymes in fresh and cultured leukemia-lymphoma cells. III. Esterase isoenzyme in monocytes.

The expression of a particular alpha-naphthyl acetate esterase isoenzyme which is specific for monocytes was examined in a panel of cultured leukemia-lymphoma cell lines (n = 88), freshly obtained leukemia-lymphoma cells (n = 527), and in fresh (n = 10) and cultured (n = 22) leukemia cells treated with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). The sodium fluoride-sensitive isoenzyme was separated by isoelectric focusing on horizontal thin-layer polyacrylamide gels. The esterase isoenzyme was not detected in untreated or TPA-treated lymphoid, erythroid, or Hodgkin's disease-derived cell lines, but was seen in leukemia cell lines of monocytic origin. TPA induced the new expression of this marker isoenzyme in two leukemia cell lines of promyelocytic and erythroid origin that are known to differentiate along the monocytic-macrophage cell lineage; TPA stimulation increased the staining intensity of the band in monocytoid cell lines. This esterase isoenzyme was found in 92% of the cases classified morphologically as acute myelomonocytic or monocytic leukemia, but only in 3% of the non-monocytic acute myeloid leukemias. All lymphoid or erythroid leukemias or lymphomas were negative. Treatment with TPA of AML and CML cells, which commonly differentiate to monocyte/macrophage-like cells, showed de novo the monocyte-specific isoenzyme. It is concluded that this isoenzyme is a characteristic marker for monocytic leukemia cells and will be a useful tool for the discriminatory identification of the monocytic element in normal and leukemic cells.

B-Lymphocytes↗

Nonspecific esterase of acute promyelocytic leukemia (M3).

Leukemic cells of 43 patients with acute promyelocytic leukemia (M3) were investigated morphologically and cytochemically to determine the percentage of aberrant enzymes and whether or not the presence impacts on the clinical outcome. Twelve patients (27.9%) showed alpha-naphthyl acetate esterase (ANAE) activity in their leukemic cells, and two of these cases revealed remarkably low myeloperoxidase (MPO) positivity, a pattern seen in monocytic precursors. However, further cytochemical evidence for this monocytic feature, the inhibition of naphthol AS-D acetate esterase (NASDA) activity with sodium fluoride (NASDA-F), was found in only five of these nine patients. In 31 cases (72.1%), there was minimal ANAE activity. Of interest, two of these were devoid of naphthol AS-D chloroacetate esterase (CAE), which is prominently displayed in neutrophilic granulocytes, even though these leukemic cells were 100% intensely positive for MPO activity. Between the two groups with and without ANAE activity, there were no remarkable differences in the distribution of sex and age, hematological findings, and rates of complete response. Our study has confirmed the cytochemical heterogeneity of M3 with no obvious relationship between this heterogeneity and early therapeutic outcome.

Adolescent↗

Distribution of esterase activity at the level of the epithelium of the diffusely infiltrated area (DIA) and of the cloaca in the Gallus domesticus: an ultrastructural study.

Esterase activity studies on the areas of lymphoid infiltration in the bursa of Fabricius (diffusely infiltrated area) and the dorsal wall of the cloaca showed that the epithelial cells exhibit varying degrees of diffuse esterase activity; it was possible to demonstrate the presence of spot-like esterase positivity in star-shaped cells in the epithelium itself. These cells can be found at various levels from the proximal to the distal region and have also been observed in the connective tissue of the tunica propria; as a result, the hypothesis has been advanced that they are connective tissue.

Animals↗

In vitro hydrolysis of RR,SS-threo-methylphenidate by blood esterases--differential and enantioselective interspecies variability.

Enantioselective in vitro hydrolysis of methylphenidate (MPH) by the blood esterases of seven mammalian species is reported. The species included rats, rabbits, dogs, cattle, horses, monkeys, and humans. In vitro incubations up to 8 h were carried out in plasma, red blood cells, and whole blood of the various species. Enantioselective differences were evident among the different species on comparison of the data obtained from the three biological fluids. The esterases present in plasma appeared to show greater activity in the hydrolysis of MPH in all species where comparison with the other two biofluids was possible. Only in the case of humans did esterases present in plasma and red blood cells demonstrate opposite enantioselectivity in the hydrolysis of MPH. Thus after 8 h incubation, the RR-MPH/SS-MPH ratios in plasma and red blood cells were 0.31 and 1.16, respectively.

Animals↗

Intracellular pH, esterase activity, and DNA measurements of human lung carcinomas by flow cytometry.

An important intention of flow cytometric investigations is to obtain biochemical and biophysical information about cells which is suitable for automated tumor diagnosis. In this study, the ploidy status, the intracellular pH value, the intracellular esterase activity, and the cell volume of vital cells and the DNA and cell volume of dead cells were measured in cancerous tissue and normal lung tissue of 30 patients by flow cytometry. The cell samples were simultaneously stained with the pH and esterase indicator dye 1.4-diacetoxy-2,3-dicyanobenzene (ADB) and propidium iodide (PI). The flow cytometric measurements were performed in three-parameter list mode. The data were evaluated on an AT-compatible personal computer with the DIAGNOS1 program system for automated diagnosis of flow cytometric list mode data. Significant differences were found between normal and malignant tissue in DNA ploidy, in the intracellular esterase activity, in the cell, volume and in the percentage of inflammatory cells and parameters of necrosis. DNA-aneuploidy was observed in 38% of the lung carcinomas. The simultaneous detection of DNA-aneuploidy and tumor-associated properties in a multifactorial analysis led to correct automatic tumor diagnosis in 85% of cases. DNA-aneuploidy was found at a significant higher frequency in advanced tumors. Adenocarcinomas displayed DNA-aneuploidy more often (80%) than squamous cell carcinomas (33%).

Adenocarcinoma↗

Effect of esterase on methacrylates and methacrylate polymers in an enzyme simulator for biodurability and biocompatibility testing.

Current in vitro biocompatibility methods do not evaluate the degradation of biomaterials after contact with enzymes that might be present in the oral or systemic environment. In this study, two methods of in vitro enzyme degradation and a method for the separation of the degradative products by high performance thin-layer chromatography (HPTLC) are reported. In the first method two dental adhesives, Scotchbond and Scotchbond II, and two dental composites, Heliomolar and P-50, were evaluated. These materials were incubated with four different enzymatic preparations for periods of up to 72 h. The enzymes were lipase, esterase, and liver enzyme extracts from both mouse and rat. Chloroform soluble products extracted from the aqueous phase were examined by HPTLC for decomposition products resulting from enzyme activity. The second method was similar, but analyzed the aqueous fraction directly without chloroform extraction. In this method five dental restorative materials, P-50, P-30, Scotchbond II, Silux, and Silux Plus, were incubated with a nonspecific porcine liver esterase. In addition to the polymerized biomaterials, monomers containing methacrylic acid units were also hydrolyzed with esterase and analyzed by ion chromatography to establish the sensitivity of the enzyme simulator. Each biomaterial presented thin-layer zones not present before enzymatic action. These experiments provide support that aqueous enzymatic action may facilitate the hydrolytic weakening of polymeric biomaterials.

Animals↗

[Physiology and biochemistry of streptomycetes. VIII. Esterase activity and production of turimycin in cultures of Streptomyces hygroscopicus JA 6599].

Esterase in cell-free extracts of Streptomyces hygroscopicus JA 6599 has a temperature-optimum of 35 degrees C, a pH-optimum with p-nitrophenylacetate as substrate at pH 7.7--8.1, with alpha-naphthylacetate at pH 7--9. Michaelis constants in cell-free extracts: with alpha-naphthylacetate Km = = 0.71 mM, with p-nitrophenylacetate Km = 0.21 mM. Phenylesters were better hydrolyzed than naphthylesters, phenylacetate was best hydrolyzed; beta-naphthylacetate was better hydrolyzed than alpha-naphthylacetate. Among the naphthylesters the ester of propionic acid was hydrolyzed best. Caprylate, stearate, and 0,0-diethyl-0-(p-nitrophenyl)-phosphate inhibit the splitting of alpha-naphthylacetate. A comparison with esterases of other biological origin shows that the enzyme studied can be a carboxylesterase (E.C.3.1.1.1.). In cultures of JA 6599 V13 and JA 6599-6 the change of esterase activity during the fermentation was determined. We found a carrelation between the enzymatic activity and the antibiotic-concentration in the culture medium.

Acetates↗

Physical model evaluation of topical prodrug delivery--simultaneous transport and bioconversion of vidarabine-5'-valerate IV: Distribution of esterase and deaminase enzymes in hairless mouse skin.

A semiquantitative assessment of estrase and deaminase distributions in hairless mouse skin was performed in vitro. The enzyme activities were quantified using 3H-vidarabine and tis 5'-valerate as the substrates. Full-thickness skin of the hairless mouse was cut into two halves, and each half was homogenized in pH 7.4 buffer. BQOTH THE SUPERNATE AND THE RESIDUE OF THE HOMOGENATE were assayed for esterase and deaminase activities. Results show that the outer half-thickness of the skin contained more esterase but slightly less deaminase than the other half. The characteristics of the esterase and the deaminase reactions also were studied employing the crude enzyme extract; these reactions were essentially irreversible. The deaminase reaction was in the linear region of Michaelis-Menten kinetics for substrate concentrations up to 4.5 x 10(-5) M.

Aminohydrolases↗

Identification of serine esterases in tissue homogenates.

Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP. The nature of the enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and alpha-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity.

Acetylcholinesterase↗

Role of cysteine residues in esterase from Bacillus stearothermophilus and increasing its thermostability by the replacement of cysteines.

Bacillus stearothermophilus esterase contains two free cysteine residues at positions of 45 and 115, which react with sulfhydryl reagents resulting in a significant decrease in the enzymatic activity. To understand the role of the cysteine residues in catalytic regions of the esterase, the residues were replaced with serine or alanine by site-directed mutagenesis to construct four single-mutated enzymes (C45A, C45S, C115A, C115S) and two double-mutated ones (C45/115A and C45/115S). Wild-type and mutant enzymes were produced in Escherichia coli cells and purified to homogeneity to examine their chemical and kinetic properties. These mutant enzymes had esterase activity, which suggested that none of the cysteines were required for its activity. Moreover, replacement of both two-cysteine residues made the enzyme insensitive to p-chloromercuribenzoic acid and extensively stabilized it at high temperatures of around 70 degrees C. These results demonstrate that replacement of free cysteine residues by site-directed mutagenesis can improve the thermostability of thermophilic enzymes.

Base Sequence↗

Plasma esterase activities in rats fed magnesium-deficient diets.

In a study with rats it was determined whether dietary magnesium concentration affects plasma esterase activities. The feeding of a diet with 0.01% (w/w) instead of 0.04% magnesium reduced plasma magnesium concentration by 50%. Plasma total esterase, arylesterase and butyrylcholinesterase activities were significantly decreased in the magnesium-deficient rats. In rats fed a diet containing 0.02% magnesium, plasma magnesium concentration was lowered by 30%, and group mean plasma total esterase activity was decreased, but not the activities of arylesterase and butyrylcholinesterase.

Animals↗

Hexachlorobenzene residues and effects on esterase activities in pre-weanling rats after a reciprocal transfer between HCB-treated and control dams.

Female Wistar rats were divided into two groups. One group was fed ad libitum powdered rat chow containing 80 ppm hexachlorobenzene (HCB) and the other was fed HCB-free diet 2 weeks before mating until the termination of the experiment. Immediately after parturition, the pups were randomly culled to 10 per litter and 5 pups were reciprocally transferred between HCB and control dams. All pups within same paired litters were sacrificed on the 16th to 17th day and on the 20th to 23rd day after birth. Liver esterase activity toward indophenyl, thiophenyl, and p-nitrophenyl acetates was determined. The liver to body weight ratio, liver esterase activity and HCB residues were significantly higher in pups nursed by HCB-fed dams than in those nursed by control dams. The HCB residue in the liver, kidney, spleen, heart and brain was analyzed by gas-liquid chromatography. At about the weaning time, the HCB residue levels in pups transferred after parturition from HCB fed to control dams were similar to those in pups which remained with the control dams. The results demonstrated that HCB in tissues at about weaning time was obtained largely through milk. In addition, transmission of HCB through milk had greater effects on esterase activities in suckling pups than placental transmission. The pups born to HCB-treated dams and nursed by the control dams were similar to the pups born and nursed by the same control dams.

Animals↗

Isoenzyme status and genetic variability of serum esterases in the lesser snow goose, Anser caerulescens caerulescens.

A maximum of 22 bands comprising four esterase subgroups--acetylesterase, carboxylesterase, cholinesterase, and acetylcholinesterase--were detected following electrophoresis of lesser snow goose sera on polyacrylamide gels. A minimum of seven structural genes was surmised to be involved in the biosynthesis of these enzymes following physiochemical characterizations. The genetic variability of these loci was calculated to be 1.25% average heterozygosity, while 14.3% of the loci were polymorphic. These estimates of genetic variability were substantially lower than those reported for other vertebrate species. The low degree of genetic variability found in snow goose serum esterases coupled with the extensive protein multiplicity observed may possibly reflect an adaptive strategy based on "biochemical plasticity" rather than genic heterozygosity for this species. The nature of evolutionary forces acting upon multiple enzyme systems such as esterases is discussed. The concept of "conditional neutrality" is introduced and defined within this context.

Animals↗

Esterases of Mus musculus: substrate and inhibition characteristics, new isozymes, and homologies with man.

A wide range of fluorogenic and naphthol esters has been tested as substrates for mouse esterases. New esterases have been identified in liver and kidney extracts with palmityl, oleyl, and elaidyl esters. From substrate, inhibition, and molecular weight studies, three homologies between human and mouse esterases are suggested. A new allele at Es-6 is also described.

4-Nitrophenylphosphatase↗

Further genetic variation at the esterase loci of Drosophila virilis.

Reexamination of the electrophoretic mobilities of esterases encoded by the Est-alpha and the Est-beta alleles of Drosophila virilis was carried out in detail using both thin-layer agar gel and polyacrylamide slab gel electrophoresis. Many allelic products with fine differences in their electrophoretic mobilities were found and designated by a new system. Some esterases separable by the agar gel method were indistinguishable using the polyacrylamide gel method. But the polyacrylamide gel method uncovered two multiband homozygotes, alpha(d).77 and beta(d) 1.28. Some allelic frequencies on the basis of the new designation were estimated in two natural populations. As a result, it is proposed that the total scope of allelic variation at the two esterase loci of Drosophila virilis is composed of discrete distribution patterns of gene frequencies, each histogram of which shows a bell-shaped pattern.

Alleles↗

Linkage analyses among five esterase loci in the laboratory rat (Rattus norvegicus).

A cluster of esterase loci has been identified on a segment of a rat linkage group V; however, the linear order of all the loci has not been established. We estimated the recombination frequencies of two locus combinations among five esterase loci (Es-1, Es-2, Es-3, Es-4, and Es-Si) and the linear order of the loci by using three sets of backcross matings: (1) (K:W x IS) x IS, (2) (K:W x IS) x IS, and (3) (SHR x W) x W). The linear order was determined to be ES-1-ES-4-ES-2-ES-3-ES-Si, although the order of ES-2 and Es-4 remains tentative. The sex-influenced esterase (Es-Si) was demonstrated to be distinct from Es-1 and was proposed to be Es-Si locus with two alleles of Es-Si(a) (positive) and Es-Si(b) (null).

Alleles↗