Enzymatic deacylations of esterified saccharides--III. Comparison of de-esterifications by serum esterases from different sources

1. 14C-labelled methyl 2,6-di-O-pivaloyl-alpha-D-glucopyranoside (1) was used as a substrate for esterases from rabbit, guinea pig, mouse, donkey, pig, horse, sheep and human sera. 2. Stepwise de-esterification of the diester substrate 1 occurred with rabbit, guinea pig and mouse serum. Data on time-course experiments and kinetic data are reported. 3. The use of donkey, pig, horse, sheep and human serum led to the migration of the 2-O-pivaloyl group in substrate 1 to the position 4- in the sugar molecule, followed by stepwise de-esterifications of both 1 and the newly formed methyl 4,6-di-O-pivaloyl-alpha-D-glucopyranoside (4). A report is given on the time-course experiments.     

Analysis of different de-esterification mechanisms for pectin by enzymatic fingerprinting using endopectin lyase and endopolygalacturonase II from A. niger

A series of pectins with different distribution patterns of methyl ester groups was produced by treatment with either plant (p-PME) or fungal pectin methyl esterases (f-PME) and compared with those obtained by base catalysed de-esterification. The products generated by digestion of these pectins with either endopectin lyase (PL) or endopolygalacturonase II (PG II) from Aspergillus niger were analysed using matrix assisted laser desorption ionisation mass spectrometry (MALDIMS) and high-performance anion-exchange chromatography with pulsed amperometric or UV detection (HPAEC-PAD/UV). Time course analysis using MALDIMS was used to identify the most preferred substrate for each enzyme. For PL, this was shown to be fully methyl esterified HG whereas for PG II, long regions of HG without any methyl esterification, as produced by p-PME was the optimal substrate. The blockwise de-esterification caused by p-PME treatment gave a decrease of partly methylated oligomers in PL fingerprints, which did not effect the relative composition of partly methylated oligomers. PG II fingerprints showed a constant increase of monomers and oligomers without any methyl ester groups with decreasing degree of esterification (DE), but almost no change in the concentration of partly methylated compounds. PL fingerprints of f-PME and chemically treated pectins showed decreasing amounts of partly methyl esterified oligomers with decreasing DE, together with a relative shift towards longer oligomers. PG II fingerprints were characterised by an increase of partly methylated and not methylated oligomers with decreasing DE. But differences were also seen between these two forms of homogenous de-esterification. Introduction of a certain pattern of methyl ester distribution caused by selective removal of certain methyl ester groups by f-PME is the most reasonable explanation for the detected differences.     

Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum.

The four most important non-specific carboxylesterases from rat liver were assayed for their ability to hydrolyse retinyl esters. Only the esterases with pI 6.2 and 6.4 (= esterase ES-4) are able to hydrolyse retinyl palmitate. Their specific activities strongly depend on the emulsifier used (maximum rate: 440 nmol of retinol liberated/h per mg of esterase). Beside retinyl palmitate, these esterases cleave palmitoyl-CoA and monoacylglycerols with much higher rates, as well as certain drugs (e.g. aspirin and propanidid). However, no transacylation between palmitoyl-CoA and retinol occurs. Retinyl acetate also is a substrate for the above esterases and for another one with pI 5.6 (= esterase ES-3). Again the emulsifier influences the hydrolysis by these esterases (maximum rates: 475 nmol/h per mg for ES-4 and 200 nmol/h per mg for ES-3). Differential centrifugation of rat liver homogenate reveals that retinyl palmitate hydrolase activity is highly enriched in the plasma membranes, but only moderately so in the endoplasmic reticulum, where the investigated esterases are located. Since the latter activity can be largely inhibited with the selective esterase inhibitor bis-(4-nitrophenyl) phosphate, it is concluded that the esterases with pI 6.2 and 6.4 (ES-4) represent the main retinyl palmitate hydrolase of rat liver endoplasmic reticulum. In view of this cellular localization, the enzyme could possibly be involved in the mobilization of retinol from the vitamin A esters stored in the liver. However, preliminary experiments in vivo have failed to demonstrate such a biological function.     

Distribution of methoxyl groups in apple pectic substances

The distribution of methoxyl groups in apple pectic substances was investigated by means of fractionation on ion-exchange and gel-filtration columns and by means of degradation of pectin fractions by pectin lyase and pectate lyase. Pectin fragments thus obtained were fractionated by gel-permeation chromatography and high-pressure liquid chromatography. It was concluded that a heterogeneous intermolecular distribution of the methoxyl groups exists with peaks at degrees of esterification of about 50%, 70% and 95%. The intramolecular distribution of the methoxyl groups cannot be distinguished from a random distribution. Since plant pectin esterases cause a blockwise de-esterification, it is unlikely that the biosynthesis of apple pectic substances passes through a stage of 100% esterification after which partial de-esterification by pectin esterase occurs.     

Initial degradation of dimethylphthalate by esterases from Bacillus species

Dimethylphthalate (DMP), one of the phthalate esters, is used in the manufacture of plasticizers, insect repellents, and synthetic fibers, and contributes to environmental pollution. In the present study, we report a novel bacterium belonging to the Bacillus sp., which has the ability to utilize DMP as the sole source of carbon. The esterases from the cell-free extract of the Bacillus de-esterified DMP. Native polyacrylamide gel electrophoresis showed the presence of four isoesterases designated Et1--4. The isoesterases Et-4 and Et-1 showed a higher preference towards DMP hydrolysis as compared with Et-2 and 3. A megaplasmid of about 60 kb was detected in this bacterium. The ability of this bacterium to utilize DMP as the sole source of carbon was lost upon plasmid curing. The isoesterases Et-1--4 were absent in the cell-free extracts of the cured bacterium. The results from our studies clearly demonstrate that de-esterification is the initial step in the degradation of DMP and the genes for these esterases seem to be harbored on the plasmid in this bacterium.     

Processing of the phosphorylated recognition marker in lysosomal enzymes. Characterization and partial purification of a microsomal alpha-N-acetylglucosaminyl phosphodiesterase

N-Acetylglucosamine(1)phospho(6)mannose groups recently identified in lysosomal enzymes were proposed to be precursors of the recognition markers terminating with mannose 6-phosphate (Tabas, I., and Kornfeld, S. (1980) J. Biol. Chem. 225, 6633-6639; Hasilik, A., Klein, U., Waheed, A., Strecker, G., and von Figura, K. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 7074-7078). To study the presumptive enzyme removing N-acetylglucosamine from the diester, an assay was developed using a radioactive oligosaccharide containing diester groups of the above structure. An alpha-N-acetylglucosaminyl phosphodiesterase cleaving this substrate in vitro was found in human placenta and in rat liver. The enzyme was solubilized from the microsomal fraction of human placenta and more than 800-fold purified with 75% yield. It is distinct from the lysosomal alpha-N-acetylglucosaminidase by the criteria of immunological cross-reactivity, substrate specificity, and heat stability. The partially purified enzyme cleaves alpha-N-acetylglucosamine phosphodiester bonds in oligosaccharides from lysosomal enzymes, in lysosomal enzymes, and in UDP-N-acetylglucosamine. We propose that the microsomal alpha-N-acetylglucosaminyl phosphodiesterase is involved in the processing of the phosphorylated recognition marker in lysosomal enzymes.     

Extracellular esterase activity as an indicator of endoplasmic reticulum calcium depletion

Abstract

Context: Endoplasmic reticulum (ER) calcium depletion is associated with diverse diseases, including cardiac, hepatic, and neurologic diseases.

Objective: The aim of the present study was to identify and characterize an endogenous protein that could be used to monitor ER calcium depletion comparably to a previously described exogenous reporter protein.

Materials and methods: The use of a selective esterase-fluorescein diester pair allowed for carboxylesterase activity in extracellular fluid to be measured using a fluorescent readout. Cell culture media from three different cell lines, rat plasma, and human serum all possess quantifiable amounts of esterase activity.

Results: Fluorescence produced by the interaction of carboxylesterases with a fluorescein diester substrate tracks with pharmacological and physiological inducers of ER calcium depletion. The fluorescence measured for in vitro and in vivo samples were consistent with ER calcium depletion being the trigger for increased esterase activity.

Discussion: Decreased luminal ER calcium causes ER resident esterases to be released from the cell, and, when assessed concurrently with other disease biomarkers, these esterases may provide insight into the role of ER calcium homeostasis in human diseases.

Conclusion: Our results indicate that carboxylesterases are putative markers of ER calcium dysfunction.     

Modulation of the degree and pattern of methyl-esterification of pectic homogalacturonan in plant cell walls. Implications for pectin methyl esterase action, matrix properties, and cell adhesion

Homogalacturonan (HG) is a multifunctional pectic polysaccharide of the primary cell wall matrix of all land plants. HG is thought to be deposited in cell walls in a highly methyl-esterified form but can be subsequently de-esterified by wall-based pectin methyl esterases (PMEs) that have the capacity to remove methyl ester groups from HG. Plant PMEs typically occur in multigene families/isoforms, but the precise details of the functions of PMEs are far from clear. Most are thought to act in a processive or blockwise fashion resulting in domains of contiguous de-esterified galacturonic acid residues. Such de-esterified blocks of HG can be cross-linked by calcium resulting in gel formation and can contribute to intercellular adhesion. We demonstrate that, in addition to blockwise de-esterification, HG with a non-blockwise distribution of methyl esters is also an abundant feature of HG in primary plant cell walls. A partially methyl-esterified epitope of HG that is generated in greatest abundance by non-blockwise de-esterification is spatially regulated within the cell wall matrix and occurs at points of cell separation at intercellular spaces in parenchymatous tissues of pea and other angiosperms. Analysis of the properties of calcium-mediated gels formed from pectins containing HG domains with differing degrees and patterns of methyl-esterification indicated that HG with a non-blockwise pattern of methyl ester group distribution is likely to contribute distinct mechanical and porosity properties to the cell wall matrix. These findings have important implications for our understanding of both the action of pectin methyl esterases on matrix properties and mechanisms of intercellular adhesion and its loss in plants.     

Genetic control of liver esterase forms in chickens

Six regions of esterase activity designated I to VI were resolved from liver extracts of chickens by horizontal starch gel electrophoresis. These esterases were further characterized on the basis of their substrate affinities and differential responses to various inhibitors.
Genetic variation was found in esterases of region VI which appeared to be ali-esterase. Four phenotypes, A, B, AB and O, were observed. These phenotypes were shown to be controlled by one autosomal locus, designated Es-3 , with alleles Es-3 ^A, Es-3 ^B and Es-3 ^O. This locus is not closely linked to the blood group loci A and B , serum alkaline phosphatase ( Ap ), liver acid phosphatase ( Acp-2 ) and serum esterase ( Es-1 ) loci.     

A method for the estimation of esterase synthesis and degradation and its application to evaluate the influence of insulin and glucagon.

The irreversible reaction between liver esterases and the active-site-directed inhibitor bis(4-nitrophenyl)phosphate can be used in vivo both for the estimation of the esterase contents and for the measurement of the esterase degradation rates. A method based on this reaction is described which allows the simultaneous estimation of the rate constants of degradation and synthesis of esterases during a period of change in protein concentration. Rat liver was found to contain about 1 mg of organophosphate-binding esterases per g of fresh tissue while the microsomal fraction contains about 30 mg of esterases per g of microsomal protein. Esterase degradation and de novo synthesis were shown to remain in equilibrium for a period of at least five days following the injection of 10 mg bis(4-nitro-[14C]phenyl)phosphate per kg. The decrease of the relative amount of labeled esterases with time was found to follow first-order kinetics yielding an average esterase degrading constant of 0.0165 h-1 which corresponds to a half-life of 42 h. These data were confirmed by an independent experiment using one of the standard procedures for the estimation of degradation rates: [14C]leucine was incorporated and one of the esterases was subsequently isolated by immuno-precipitation. Using isoelectric focussing and dodecyl sulfate electrophoretic methods, the various esterase isoenzymes appeared to have very similar, if not identical turnover rates. This method for the estimation of the turnover characteristics was applied to evaluate hormone effects on liver esterases. The time course of the contents and the turnover of liver esterases was measured under the influence of glucagon treatment in diabetic rats and under the influence of high doses of insulin. The esterase content decreased faster than the average content of microsomal protein under the influence of glucagon. The reverse effect was observed with insulin-treated rats. Both insulin and glucagon apparently reduced the intracellular esterase turnover in rat liver. Kinetic analysis of the results revealed that insulin mainly lowered the esterase degradation rate, though the rate of esterase synthesis might also have been restricted. In the glucagon-treated rats the de novo synthesis of esterases was strongly reduced.     

Purification and characterization of two thermostable acetyl xylan esterases from Thermoanaerobacterium sp. strain JW/SL-YS485.

Two acetyl esterases (EC 3.1.1.6) were purified to gel electrophoretic homogeneity from Thermoanaerobacterium sp. strain JW/SL-YS485, an anaerobic, thermophilic endospore former which is able to utilize various substituted xylans for growth. Both enzymes released acetic acid from chemically acetylated larch xylan. Acetyl xylan esterases I and II had molecular masses of 195 and 106 kDa, respectively, with subunits of 32 kDa (esterase I) and 26 kDa (esterase II). The isoelectric points were 4.2 and 4.3, respectively. As determined by a 2-min assay with 4-methylumbelliferyl acetate as the substrate, the optimal activity of acetyl xylan esterases I and II occurred at pH 7.0 and 80 degrees C and at pH 7.5 and 84 degrees C, respectively. Km values of 0.45 and 0.52 mM 4-methylumbelliferyl acetate were observed for acetyl xylan esterases I and II, respectively. At pH 7.0, the temperatures for the 1-h half-lives for acetyl xylan esterases I and II were 75 degrees and slightly above 100 degrees C, respectively.     

Acinar primary cell culture from the digestive gland of Pecten maximus (L.): an original model for ecotoxicological purposes

The purpose of this study was to establish an original primary acinar cell culture model for the mollusc bivalve Pecten maximus (L.), and to define its values and limits for subsequent ecotoxicological applications. To prevent microbial contaminations occurring frequently in invertebrate cell cultures, a perfusion of the stomach-digestive gland complex was performed in situ using a sterile salt solution containing broad-range antibiotics. Digestive acini were isolated using a pronase enzyme that was removed by several washings of the acinar suspension, after which their viability and functionality were determined by three different assays: fluorescein diacetate (FDA) de-esterification, 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium (MTT) reduction and neutral red (NR) incorporation describing de-esterification, mitochondrial dehydrogenase and lysosome activity, respectively. The kinetic conditions for these assays were defined beforehand. The results showed that digestive acini could be maintained in vitro both cytologically and functionally for at least 96 h, which is sufficient for many ecotoxicological applications. Preliminary contamination assays, according to the function studied (cell esterases, mitochondrial respiration, lysosomal incorporation), indicated that polycyclic aromatic hydrocarbons had a negative effect on the survival of acini in vitro.     

Lipase hydrolysis of mammalian long-chain 1,2-alkanediol diesters. Nonrandom distribution of fatty acids

Long-chain 1,2-alkanediol diesters were isolated from the total surface lipids of golden Syrian hamsters and Swiss albino mice. Hydrolysis of the diol diester waxes with exocellular lipase from Rhizopus arrhizus delemar or with purified porcine pancreatic lipase produced free fatty acids and 2-acyl diols in about 60--80% yield. Nonrandom distribution of the constituent fatty acids at positions 1 and 2 of the alkanediols was observed. In the diester waxes from the hamster, both straight-chain and branched-chain fatty acids of 14 to 20 carbon atoms predominated at position 1 and those of 22 to 26 carbon atoms at position 2. In contrast, the diester waxes of the mouse contained mainly fatty acids of less than 19 carbon atoms, both saturated and monounsaturated, at position 2 and those of greater chain length (20 to 24 carbon atoms) at position 1. The results of the lipase hydrolysis were confirmed by degradation of the diester waxes with Grignard reagent.     

Esterase and lipase activity in Jatropha curcas L. seeds

Two new esterases (JEA and JEB) and a lipase (JL) were extracted from the seeds of Jatropha curas L. Lipase activity was only found during germination of the seeds and increased to a maximum after 4 days of germination. All enzymes were found to be most active in the alkaline range at around pH 8 and the purified (fractionated precipitation with ethanol and gel filtration) esterases were very stable at high temperatures. The molecular weight (SDS-PAGE) of both esterases was determined to be 21.6-23.5 kDa (JEA) and 30.2 kDa (JEB) and the isoelectric point was 5.7-6.1 for esterase JEA and 9.0 for esterase JEB. Most ions caused a negative influence on the activity of both esterases. Using p-nitrophenyl butyrate as a substrate JEA showed a K(m) of 0.02 mM and a v(max) of 0.26 micromol mg(-1) min(-1). Under the same conditions JEB showed a K(m) of 0.07 mM and a v(max) of 0.24 micromol mg(-1) min(-1). Both esterases hydrolyzed tributyrin, nitrophenyl esters up to a chain length of =C4 and naphtylesters up to a chain length =C6. In transesterification reactions, JL was found to be most active at very low water activities (0.2) and in high water activities, the lipase hydrolysed triglycerides into conversions above 80%. The lipase hydrolysed both short chain and long chain triglycerides at about the same rate but was inactive on alpha-methylbenzyl acetate. JL is a potentially useful biocatalyst in the hydrolysis of triglycerides in organic solvents.     

Qualitative alterations in plasma esterases in BALB/c mice following the administration of diethylnitrosamine.

Selected non-specific plasma esterases in female BALB/c mice, separated by polyacrylamide gel electrophoresis, were qualitatively altered following treatment of the mice with diethylnitrosamine (DEN). These alterations include a decreased preference for naphthyl butyrate as a substrate relative to naphthyl acetate; decreased sensitivity to enhancement by divalent cations such as Ca2+, Mg2+, and Mn2; and an increased sensitivity to inhibition by eserine. All esterase species affected were also quantitatively altered and some were testosterone-dependent.     

Enantioselective hydrolysis of oxazepam 3-acetate by esterases in human and rat liver microsomes and rat brain S9 fraction

Rates of hydrolysis of racemic and enantiomeric oxazepam 3-acetates (OXA) by esterases in human and rat liver microsomes and rat brain S9 fraction were compared. When rac-OXA was the substrate, esterases in human and rat liver microsomes were highly enantioselective toward (R)-OXA. In contrast, esterases in rat brain S9 fraction were highly enantioselective toward (S)-OXA. Hydrolysis rates of rac-OXA were highly dependent on the amount of esterases used. At 0.05 mg protein equivalent of esterases and 150 nmol of rac-OXA per ml of incubation mixture, the (R)-OXA was hydrolyzed 3.6-fold and 18.5-fold faster than (S)-OXA by rat and human liver microsomes, respectively. The specific activities (nmol of OXA hydrolyzed/mg microsomal protein/min) of liver microsomes in the hydrolysis of enantiomerically pure (R)-OXA were approximately 120 (rat) and 1,980 (human), and in the hydrolysis of enantiomerically pure (S)-OXA were 4 (rat) and 7 (human), respectively. In the incubation of rac-OXA with rat brain S9 fraction, (S)-OXA was hydrolyzed approximately 6-fold faster than (R)-OXA. Results also indicated an enantiomeric interaction in the hydrolysis of rac-OXA by esterases in rat and human liver microsomes; the presence of (R)-OXA stimulated the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA inhibited the hydrolysis of (R)-OXA. In rat brain S9 fraction, the presence of (R)-OXA inhibited the hydrolysis of (S)-OXA, whereas the presence of (S)-OXA appeared to have stimulated the hydrolysis of (R)-OXA.     

Lipoprotein substrates of lipoprotein lipase and hepatic triacylglycerol lipase from human post-heparin plasma.

The number and the substrate specificities of glutathione thiol esterases of human red blood cells have been investigated by gel electrophoresis and isoelectric focusing and staining methods devised for the location of these enzymes on gels. Several glutathione thiol esterase forms, both unspecific (with respect to the S-acyl group of the substrate) and specific were found. Electrophoresis on both polyacrylamide and agarose gels resolved three enzyme components with apparently similar substrate specificity. Isoelectric focusing in liquid column separated two unspecific thiol esterase components with S-lactoylglutathione (pI = 8.4) and S-propionylglutathione (pI = 8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (pI = 5.2) and S-succinylglutathione hydrolase (pI = 9.0). Isoelectric focusing on polyacrylamide gel resolved nine unspecific glutathione thiol esterase bands (between pH values 7.0 and 8.4). Partially purified glyoxalase II (S-2-hydroxyacylglutathione hydrolase, EC 3.1.2.6) from erythrocytes or liver still gave three components on electrophoresis and several activity bands on gel electrofocusing. These results indicate that human red cells contain at least four separate glutathione thiol esterases. Glyoxalase II, one of these enzymes, apparently occurs in multiple forms. These were neither influenced by preptreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.     

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.This work was supported by the Medical Research Council.     

Recombinant porcine intestinal carboxylesterase: cloning from the pig liver esterase gene by site-directed mutagenesis, functional expression and characterization

It was shown recently that proline-beta-naphthylamidase from pig liver resembles the gamma-subunit of pig liver esterase (PLE), which could be functionally expressed in the yeast Pichia pastoris in recombinant form (rPLE). The gene encoding rPLE shares 97% identity with the published nucleotide sequence of porcine intestinal carboxylesterase (PICE). By site-directed mutagenesis, 22 nucleotides encoding 17 amino acids were exchanged stepwise from the PLE gene yielding the recombinant PICE sequence and eight intermediate mutants. All esterases were successfully produced in P.pastoris as extracellular proteins with specific activities ranging from 4 to 377 U/mg and V(max)/K(m) values from 12 to 1000 l min(-1) x 10(-3) using p-nitrophenyl acetate as substrate. Activity-staining of native polyacrylamide gels followed by molecular mass determination suggests that the most active forms of all variants are present as trimers with a molecular mass of 190-210 kDa. All enzymes exhibit the highest activity in the pH range 8-9 and between 60 and 70 degrees C. Almost all esterases show a higher ratio of methyl butyrate hydrolase activity to proline-beta-naphthylamidase activity than rPLE.     

Metabolism of Catecholamine Esters by Cultured Bovine Brain Microvessel Endothelial Cells

The metabolism of epinine (N-methyldopamine) and epinine diesters (acetyl, benzoyl, pivaloyl, and isobutyryl) by brain endothelium was investigated using primary cultures of bovine brain microvessel endothelial cells. 3,4-Dihydroxyphenylacetic acid (DOPAC), the product of monoamine oxidase (MAO)-mediated degradation of epinine, was the only metabolite detected by HPLC with electrochemical detection following incubation of the cell monolayers with epinine or its esters. This metabolism could be inhibited by the MAO inhibitors pargyline, clorgyline, and deprenyl, with the system being most sensitive to inhibition by clorgyline. Compared with epinine, incubation of cell monolayers with the diester prodrugs led to increased drug (epinine plus epinine diesters) tissue levels. With the exception of the diacetyl ester, lower levels of DOPAC were observed with the diester prodrugs than with the parent compound. Hydrolysis by serine-dependent esterases appears to be necessary for the subsequent oxidation by MAO. The permeabilities of epinine and the diester prodrugs through endothelial cell monolayers grown on porous supports were related to their lipophilicity and molecular weight.