Esterase Components Research Articles

Butyrylcholinesterase identification in a phenylvalerate esterase-enriched fraction sensitive to low mipafox concentrations in chicken brain.

Multiple epidemiological and experimental studies have demonstrated that exposure to organophosphorus compounds (OPs) is associated with a variety of neurological disorders. Some of these exposure symptoms cannot be precisely correlated with known molecular targets and mechanisms of toxicity. Most of the known molecular targets of OPs fall in the protein family of serine esterases. We have shown that three esterase components in the soluble fraction of chicken brain (an animal model frequently used in OP neurotoxicity assays) can be kinetically distinguished using paraoxon, mipafox and phenylmethyl sulfonyl fluoride as inhibitors, and phenyl valerate as a substrate; we termed them Eα, Eβ and Eγ. The Eα-component, which is highly sensitive to paraoxon and mipafox and resistant to PMSF, has shown sensitivity to the substrate acetylthiocholine, and to ethopropazine and iso-OMPA (specific inhibitors of butyrylcholinesterase; BChE) but not to BW 284C51 (a specific inhibitor of acetylcholinesterase; AChE). In this work, we employed a large-scale proteomic analysis B with a LC/MS/MS TripleTOF system; 259 proteins were identified in a chromatographic fractionated sample enriched in Eα activity of the chicken brain soluble fraction. Bioinformatics analysis revealed that BChE is the only candidate protein identified to be responsible for almost all the Eα activity. This study demonstrates the potential information to be gained from combining kinetic dissection with large-scale proteomics and bioinformatics analyses for identification of proteins that are targets of OP toxicity and may be involved in detoxification of phosphoryl and carbonyl esters.

Kinetic interactions of a neuropathy potentiator (phenylmethylsulfonyl fluoride) with the neuropathy target esterase and other membrane bound esterases

Phenylmethylsulfonyl fluoride (PMSF) is a protease and esterase inhibitor that causes protection, or potentiation/"promotion," of organophosphorus delayed neuropathy (OPIDN), depending on whether it is dosed before or after an inducer of delayed neuropathy, such as mipafox. The molecular target of the potentiation/promotion of OPIDN has not yet been identified. The kinetic data of phenyl valerate esterase inhibition by PMSF were obtained with membrane chicken brain fractions, the animal model and tissue in which neuropathy target esterase (NTE) was first described. Data were analyzed using a kinetic model with a multienzymatic system in which inhibition, simultaneous chemical hydrolysis of the inhibitor and "ongoing inhibition" (inhibition during the substrate reaction) were considered. Three main esterase components were discriminated: two sensitive enzymatic entities representing 44 and 41 %, with I 50 (20 min) of 35 and 198 μM at 37 °C, respectively, and a resistant fraction of 15 % of activity. The estimated constant of the chemical hydrolysis of PMSF was also calculated (kh = 0.28 min(-1)). Four esterase components were globally identified considering also previously data with paraoxon and mipafox: EPα (4-8 %), highly sensitive to paraoxon and mipafox, spontaneously reactivates after inhibition with paraoxon, and resistant to PMSF; EPβ (38-41 %), sensitive to paraoxon and PMSF, but practically resistant to mipafox, this esterase component has the kinetic characteristics expected for the PMSF potentiator target, even though paraoxon cannot be a potentiator in vivo due to high AChE inhibition; EPγ (NTE) (39-48 %), paraoxon-resistant and sensitive to the micromolar concentration of mipafox and PMSF; and EPδ (10 %), resistant to all the inhibitors assayed. This kinetic characterization study is needed for further isolation and molecular characterization studies, and these PMSF phenyl valerate esterase components will have to be considered in further studies of OPIDN promotion. A simple test for monitoring the four esterase components is proposed.

A Glutathione-Dependent Detoxification System Is Required for Formaldehyde Resistance and Optimal Survival ofNeisseria meningitidisin Biofilms

The glutathione-dependent AdhC-EstD formaldehyde detoxification system is found in eukaryotes and prokaryotes. It is established that it confers protection against formaldehyde that is produced from environmental sources or methanol metabolism. Thus, its presence in the human host-adapted bacterial pathogen Neisseria meningitidis is intriguing. This work defined the biological function of this system in the meningococcus using phenotypic analyses of mutants linked to biochemical and structural characterization of purified enzymes. We demonstrated that mutants in the adhC and/or estD were sensitive to killing by formaldehyde. Inactivation of adhC and/or estD also led to a loss of viability in biofilm communities, even in the absence of exogenous formaldehyde. Detailed biochemical and structural analyses of the esterase component demonstrated that S-formylglutathione was the only biologically relevant substrate for EstD. We further showed that an absolutely conserved cysteine residue was covalently modified by S-glutathionylation. This leads to inactivation of EstD. The results provide several conceptual innovations. They provide a new insight into formaldehyde detoxification in bacteria that do not generate formaldehyde during the catabolism of methanol. Our results also indicate that the conserved cysteine, found in all EstD enzymes from humans to microbes, is a site of enzyme regulation, probably via S-glutathionylation. The adhc-estD system protects against formaldehyde produced during endogenous metabolism.

The inhibition of the high sensitive peripheral nerve soluble esterases by mipafox: A new mathematical processing for the kinetics of inhibition of esterases by organophosphorus compounds

In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act= A 1 e − k 1 It + A 2 e − k 2 It + A R (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as A R=15.1% (step 2). The model equation with a fixed A R value was again applied (step 3) to deduce the second-order inhibition rate constants ( k 1=2.6×10 6 M −1 min −1 and k 2=0.28×10 6 M −1 min −1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of A R, k 1, and k 2, the amplitudes for the two exponential (sensitive) components ( A 1 and A 2) were re-estimated ( A 1=50.2% and A 2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 μM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.

The inhibition of the high sensitive peripheral nerve soluble esterases by mipafoxA new mathematical processing for the kinetics of inhibition of esterases by organophosphorus compounds

In the study of organophosphorus (OP) sensitive enzymes, careful discrimination of specific components within a complex multienzymatic mixture is needed. However, standard kinetic analysis gives inconsistent results (i.e., apparently different kinetic constants at different inhibitor concentration) with complex multienzymatic mixtures. A strategy is now presented to obtain consistent kinetic parameters. In the peripheral nerve, soluble carboxylesterases measured with the substrate phenylvalerate (PV) are found with extremely high sensitivity to some inhibitors. Tissue preparations were preincubated with mipafox at nanomolar concentrations (up to 100 nM) for different inhibition times (up to 180 min). Inhibition data were analyzed with model equations of one or two sensitive (exponential) components, with or without resistant components. The most complex model was %act=A1e−k1It+A2e−k2It+AR (step 1). From the curve with the highest mipafox concentration (100 nM), the amplitude for the resistant component was determined as AR=15.1% (step 2). The model equation with a fixed AR value was again applied (step 3) to deduce the second-order inhibition rate constants (k1=2.6×106 M−1 min−1 and k2=0.28×106 M−1 min−1), being conserved consistently throughout all mipafox concentrations. Finally, using fixed values of AR, k1, and k2, the amplitudes for the two exponential (sensitive) components (A1 and A2) were re-estimated (A1=50.2% and A2=34.2%). The operational process was internally validated by the close similarity with values obtained by directly fitting with a three-dimensional model equation (activity versus time and inhibitor concentration) to the same inhibition data. Carboxylesterase fractions separated by preparative chromatography showed kinetic properties consistent with the kinetically discriminated components. As practical conclusion, for routine analysis of esterases in toxicological studies, a simplified procedure using the inhibition with mipafox at 30 nM, 1 μM, and 1 mM for 30 min is suggested to discriminate the main esterase components in soluble fraction preparations.

Lamb Pregastric Esterase Catalyzed Hydrolysis of 4-Nitrophenyl-Acetate and -Dodecanoate: pH, Temperature and Bile Salt Effects

Two fractions, F2 and F3, eluted by ion exchange chromatography of the commercial extract of lamb pregastric enzyme have esterase activity against 4-nitrophenylacetate, PNPA. Preheat treatment of fraction F3 at pH 7.2, 50°C for 15 min effectively removes its lipase activity against tributyrin while leaving the esterase component relatively unaffected. The esterase components in fractions F2 and F3 and the lipase component in fraction F3 have K m values against PNPA equal to 0.96, 2.1 and 2.7 mM, respectively. When 4-nitrophenyldecanoate, PNPDe, was used as substrate, the maximum activity was reached at its critical micelle concentration, 1.6 μM, for catalysis by all three enzymes. The reactivity is dependent upon pH and p K values of 6.7 and 8.4, 6.7 and 7.5, and 7.3 were determined from the lipase and esterase components in fraction F3, and the fraction F2 esterase, respectively. The dependence upon temperature of the activities of the esterase components against PNPA were determined within the range 25-47.5°C and Arrhenius parameters have been calculated. The presence of sodium taurocholate affected the activity of each enzyme to a small and differing extent.

尿素加ポリアクリルアミドゲル等電点電気泳動法による軽種馬のPiフェノタイピング

Isoelectric focusing (IEF) using polyacrylamide gel containing urea (Urea-PAG) was used for the characterization of equine protease inhibitor (Pi) types in light breed horses to solve the following problems in conventional IEF and two-dimensional (2-D) electrophoresis: typing complexity due to high heterogeneity of Pi system and overlapping of Pi and esterase (Es) components on IEF, operational complexity and time-consumption on 2-D. The Urea-PAG IEF method had high resolution capacity, clearly distinguishing at least 10 types frequently observed in light breed horses. Es components were not detected on Urea-PAG. There was little complexity of Pi typing as shown by these results, and thus the typing procedure is simpler and more rapid than conventional IEF and 2-D. The present method should thus prove quite adequate for exact classification of Pi system in light breed horses.

Identification and phyletic distribution of the plasma esterases of some anthropoid primates

Using electrophoresis, specific staining, and selective inhibition, we examined and identified esterase components in the plasma of 13 anthropoid species: 5 hominoids, 7 cercopithecoids, and 1 platyrrhine. Most species, unlike humans, exhibited one or more arylesterase and/or carboxylesterase components as well as cholinesterase and albumin-associated arylesterase. A distinctive arylesterase probably indicates pregnancy in cercopithecoids. While some characters (such as the net charge of the cholinesterase molecule) were extremely uniform across all taxa examined, other quantitative and qualitative traits showed intra- and interspecific variation. Comparison of a cladogram with the distribution of traits shows that the least phyletically stable traits are, in general, those that also exhibit intraspecific polymorphism. One genus-specific, and therefore relatively conservative, trait (weak dimer-dimer bonding in the cholinesterase molecule) evidently appeared independently among papionin cercopithecoids and hominoids. The most parsimonious interpretation of this distribution reinforces the notion of a Pan-Gorilla-Homoclade sororally related to Pongo.In general, plasma esterases are an underexploited source of genetic markers in primates.

Enzymatic production of ethanol from cellulose using soluble cellulose acetate as an intermediate

A two-stage process for the enzymatic conversion of cellulose to ethanol is proposed as an alternative to currently incomplete and relatively slow enzymatic conversion processes employing natural insoluble cellulose. This alternative approach is designed to promote faster and more complete conversion of cellulose to fermentable sugars through the use of a homogeneous enzymatic hydrolysis reaction. Cellulose is chemically dissolved in the first stage to form water-soluble cellulose acetate (WSCA). The WSCA is then converted to ethanol in a simultaneous saccharification-fermentation with Pestal-otiopsis westerdijkii enzymes (containing cellulolytic and acetyl esterase components) and yeast.Water-soluble cellulose acetate was successfully prepared from purified wood cellulose (Solka Floe) and chemical reagents. Enzyme pretreatment of WSCAto form metabolizable sugars was a necessary step in achieving practical conversion of WSCA to ethanol using yeast. The results showed that WSCA has a low enzyme requirement and a high convertibility to reducing sugars with enzymes from P. westerdijkii fungus. Pestalotiopsis westerdijkii enzymes were found to be superior to enzymes from Trichoderma viride in producing metabolizable glucose from WSCA. The yeast utilized 55-70% of the hydrolyzate sugars that were produced by P. westerrlijkii enzymes on WSCA and produced ethanol. The acetate that was liberated into solution by the action of acetyl esterase enzymes on WSCA was found to have a stimulatory effect on ethanol production in yeast. This is an important feature that can be used to advantage in manipulating the conversion to maximize the production of ethanol. Hence, the simultaneous saccharification-fermentation of WSCA to ethanol using P. westerdijkii enzymes and yeast has features that are highly desirable for developing an economical cellulose conversion process.

Kinetics of substrate hydrolysis and inhibition by mipafox of paraoxon-preinhibited hen brain esterase activity.

For the purpose of assessing the neurotoxic potential of organophosphorus compounds, it has been determined that paraoxon-preinhibited hen brain has both neurotoxicant (mipafox)-sensitive (neurotoxic esterase; NTE) and -insensitive esterase components. Several experiments designed to investigate the kinetic parameters governing the reaction of these esterases with two substrates and one organophosphorus inhibitor are presented. First, kinetic parameters for the hydrolysis of phenyl valerate and phenyl phenylacetate were measured. At 37 degrees C, the Km values of NTE for phenyl valerate and phenyl phenylacetate were found to be about 1.4 X 10(-3) and 1.6 X 10(-4) M respectively. At 25 degrees C, the Km of NTE for phenyl valerate was determined to be about 2.4 X 10(-3) M. Secondly, the kinetic constants of NTE for mipafox were measured at both 25 degrees C and 37 degrees C. With either phenyl valerate or phenyl phenylacetate as substrate, the Km at 37 degrees C was determined to be about 1.8 X 10(-4) M, and the phosphorylation constant (k2) was about 1.1 min-1. For phenyl valerate only, the Km at 25 degrees C was found to be about 6 X 10(-4) M, and the k2 was about 0.7 min-1. The data obtained at 25 degrees C were analysed by using a two-component model without formation of Michaelis complex, a two-component model with formation of Michaelis complex on the second component (NTE), or a three-component model without formation of Michaelis complex. The fact that the Michaelis model fit the data significantly better than either of the other two models indicates that the higher apparent Ki values that occur with low concentrations of mipafox are due to formation of Michaelis complex at high concentrations, rather than because of the presence of two NTE isoenzymes, as has been suggested by other investigators.

Molecular diversity and plant disease resistance: An electrophoretic comparison of near-isogenic lines of wilt-resistant or -susceptible Pisum sativum L. cv. William Massey

The soluble proteins from two near-isogenic lines of Pisum sativum. cv. William Massey have been compared electrophoretically. These lines differ in their physiological response to wilt (Fusarium oxysporum f. sp. pisi), one being susceptible and the other resistant. The total protein profiles derived from the two lines appear to be identical. The resistant line differs electrophoretically from the susceptible line in carrying an esterase component which has been derived from its resistant parent, cultivar Delwiche Commando.

Serum esterase genetics: identification and hormone induction of the Es-1b esterase in inbred rats.

A previously unrecognized esterase from the sera of the appropriate strains of the rat Rattus norvegicus was revealed by a discontinuous polyacrylamide gel electrophoretic technique. This esterase migrated in the albumin region, whereas a previously known major albumin esterase controlled by the Es-2 locus migrated in the postalbumin region when the method was used. The new albumin esterase component which separated from the Es-2 esterase was identified as the product of the Es-1b gene. The new albumin esterase was not detectable in the sera of sexually mature males of the appropriate genotype, because the activity level of this esterase was influenced by sex hormones, especially androgen.

Glutathione thiol esterases of human red blood cells: Fractionation by gel electrophoresis and isoelectric focusing

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 (p I=8.4) and S-propionylglutathione (p I=8.1) as the best substrates, respectively, and two specific enzymes, S-formylglutathione hydrolase (p I=5.2) and S-succinylglutathione hydrolase (p I=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 pretreatment of the samples with neuraminidase or thiols nor were interconvertible during the fractionations.

Electrophoretic investigation of blood and parotoid venom proteins in Bufo americanus americanus and Bufo woodhousei fowleri

1. 1. Blood and parotoid venom proteins of Bufo americanus americanus and Bufo woodhousei fowleri were studied using vertical block polyacrylamide gel electrophoresis. 2. 2. Plasma and venom contained no components of identical electrophoretic mobility in either species. 3. 3. Venom of both soecies contained esterases; certain esterase components appeared to be species specific. 4. 4. No lipoprotein component was found in the venom of either species. 5. 5. Venom of both species contained glycoproteins. 6. 6. Use of a non-ionic detergent improved the handling of the venom. 7. 7. Wart and parotoid venom proteins were qualitatively identical. 8. 8. Plasma transferrin and albumin components of both species were polymorphic. Single and double banded transferrin and albumin phenotypes were found in both species. 9. 9. Hemoglobin of both species was found to be single banded and to have identical electrophoretic mobility. 10. 10. No seasonal or sexual variation was apparent in venom or plasma proteins of either species.

Enzyme Polymorphisms of Ideles Populations (Ahaggar, Algeria) and the Iwellemeden Kel Kummer Twaregs (Menaka, Mali)

Surveys dealing with enzyme polymorphisms have recently been conducted in the Sahara. Results from two populations are reported here: 227 inhabitants of Ideles village (Ahaggar, Algeria); 285 nomads of a genetic isolate, the Kel Kummer Twareg tribe (Menaka, Mali). The four classical molecular variants of G6PD:A+, A-, B+, B-, are found in Ideles. The frequency of the G6PD A+ Negroid variant reaches 15% in Ideles and 7.7% among the Kel Kummer. However, gene frequencies will have to be recalculated after a study of the genetic transmission through families. The PGDC gene of 6PGD is especially frequent in the Kel Kummer where 10 'Canning' phenotypes have been observed. The PGM distribution of alleles at locus 1 in Ideles is the same as in the Mediterranean populations. The pa gene of acid phosphatase, relatively frequent in Ideles, has been excluded by drift from the Kel Kummer gene pool. AK and LDH enzymes have also been studied in both samples. The abnormal Ea1 mutation of serum pseudocholinesterase exists in Ideles and in the Kel Kummer as in other populations of the Sahara; the C5 esterase component was revealed by electrophoresis in 5% of the Kel Kummer people.

Haemorrhagic snake venoms and kallikrein inhibitors as tools for the study of factors determining the integrity of the vessels wall

Hemorrhagic snake venoms contain a multitude of factors which either through a direct action or indirectly through the release of some mediator disturb the integrity of the vessel wall. For example the protease and/or esterase components in some of such venoms are enzymes known to release polypeptide kinins, which are considered as one of the mediators of microvascular functional disturbances. Previously we developed a method (4,5), which allows to study quantitatively the hemorrhagic effect of snake venoms on pulmonary surface vessels. The venom of Agkistrodon piscivorus is recognized to contain a kinin releasing component (10). We therefore examined the hemorrhagic action of this venom under the influence of some compounds which interfere with the kallikrein-kinin system. The effect of the same compounds were also studied on the hemorrhagic action of Naja naja venom, which is not known to release kinins and from which we have shown that its hemorrhagic effect is dependent on its content of a basic polypeptide (5), the latter unlikely to possess protease or esterase activity (6).

The esterases of Mortierella ramanniana in relation to taxonomy.

SUMMARY: The esterase profiles of 9 strains of Mortierella ramanniana grown on glucose and olive-oil media were determined by starch-gel electrophoresis. The enzymes produced on the oil medium were in most instances produced also on the glucose medium, but on the latter some additional esterase components were produced. The esterase patterns of the different strains varied widely; only one esterase was common to all strains grown on glucose medium, and a second esterase was common to all grown on the olive-oil medium. The data available do not suggest that esterase profiles are of taxonomic significance for these fungi.

Serum Esterase Groups of Pacific and Atlantic Tunas

Starch-gel electrophoretic analysis of serum specimens of 8201 skipjack tuna (Katsuwonus pelamis), 187 albacore (Thunnus alalunga), 107 bigeye tuna (T. obesus), and 65 yellowfin tuna (T. albacares) taken from the Pacific and Atlantic oceans demonstrated individual genetic variations in the serum esterase component within each species. The esterase system proved to be independent of any known blood group systems, of sex, and of size of fish in each species. In skipjack tuna extensive data allowed an application of the system to the problem of identifying subpopulations, or isolated breeding populations. Heterogeneity tests for the gene frequencies of the esterase system showed: a) a population in the equatorial Atlantic is different from any populations recognized in the Pacific; b) eastern Pacific (Baja California) specimens were indistinguishable from those of Hawaiian waters; and c) the western Pacific (Japan and Palau) has at least one subpopulation different from the population in Hawaiian waters.

Studies on Streptomyces griseus protease : II. The amino acid sequence around the reactive serine residue of DFP-sensitive components with esterase activity

1. 1. A highly purified esterase preparation from Streptomyces griseus protease (Pronase), active against benzoylarginine ethyl ester, was inactivated with [ 32P]DFP and hydrolysed with HCl. From the acid hydrolysate, the dipeptides Asp-[ 32P]SerP and [ 32P]SerP-Gly, as well as the tripeptide ASp-([ 32P]SerP, Gly), were isolated. This shows that the amino acid sequence around the reactive serine residue of the enzyme is Asp-Ser-Gly. 2. 2. Two other esterase components from Pronase, active against p-nitrophenl acetate, were inactivated with [ 32P]DFP and hydrolysed in the same manner. On paper electrophoresis at pH 3.5, identical patterns of 32P-labelled peptides were obtained from all three enzyme components. This indicates that the three esterases have the same amino acid sequence at the DFP-reacting site.

SERUM ESTERASES IN THE HOUSE MOUSE,MUS MUSCULUS

Recently, electrophoretically separable nonspecific esterases of the house mouse, ~Mzls rnzlsculii~s) have reckived considerable attention: Popp and Popp ( 1962), Petras ( 1963), and Ruddle and Rodericlc (1966) have described the inheritance of variants of several serum esterase components; Pelzer (1965), Biddle and Petras ( 1966), and Popp (1966) have studied differences in erythrocytic esterase zymograms; and Ruddle and Roderick (1966) have shown that the electrophoretic patterns of several Icidncy esterases are controlled by two loci. This report is concerned with the inheritance of another serum esterase variant in Mus, (preliminary report: Petras and Riddle, 1966), a new technique for better resolution of the three phenotypes controlled by locus Es-1, the relationship between Es-1-controlled serum esterases and serum albumin, and linkage data involving loci Es-1, Es-2, and the ncw locus.