The level of aryl acylamidase activity displayed by human butyrylcholinesterase depends on its molecular distribution

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.     

Comparative study of immobilized and soluble NADH:FMN-oxidoreductase-luciferase coupled enzyme system

The properties of a coupled enzyme system (NAD(P)H:FMN-oxidoreductase and luciferase) from luminous bacteria were studied. The enzymes and their substrates were immobilized in polymer gels of different types: starch (polysaccharide) and gelatin (polypeptide). Maximum activity yield (100%) was achieved with the enzymes immobilized in starch gel. An increase in K _{m app} was observed in both immobilized systems as compared with the soluble coupled enzyme system. Immobilization in starch and gelatin gels increased the resistance of the NAD(P)H:FMN-oxidoreductase and luciferase coupled enzyme system to the effects of external physical and chemical factors. The optimum pH range expanded both to the acidic and alkaline regions. The resistance to concentrated salt solutions and high temperature also increased. The coupled enzyme system immobilized in starch gel (with activation energy 30 kJ/mol) was characterized by the best thermostability. The immobilized coupled enzyme system can be used to produce a stable and highly active reagent for bioluminescent analysis.     

Texture and Microstructure of Gelatin/Corn Starch-Based Gummy Confections

Texture of gummy gels prepared with gelatin and acid modified corn starch (AMCS) was quantified by instrumental techniques and the gel microstructure was examined by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Gelatin:AMCS gummy gels were divided in two groups: Group 1, containing different gelatin (0?C10?wt%) and AMCS (0?C10?wt%) concentrations, totalizing 10?wt% solids; and Group 2, which was prepared with a fixed gelatin concentration (8?wt%) and varied AMCS concentrations (0?C5?wt%). All gummy gels were formulated with maltitol syrup and xylitol, shaped in cylindrical molds and submitted to instrumental texture profile analysis (TPA) tests and colorimetric analysis. Group 1 pure starch gels (10?% AMCS) presented the highest stringiness and adhesiveness. In samples of Group 2 the introduction of AMCS dramatically changed the structure of the gelatin gels. Thermodynamic incompatibility was evident even at the lowest AMCS concentration. Moreover, increasing AMCS concentrations lead to an increase in the number of hollow zones including starch granules inside them. In addition, introduction of AMCS in the samples of Group 2 caused an increase in hardness and opacity and a decrease in stringiness and adhesiveness. On the other hand, results from TPA tests showed that the addition of AMCS to gelatin gels in suitable proportions can be a feasible alternative in the formulation of gummy confections.     

Effect of pesticide bendiocarbamate on distribution of acetylcholine- and butyrylcholine-positive nerves in rabbit's thymus

Many pesticides used in agriculture have a negative effect on organisms. The group of hazardous pesticides includes the cholinesterase inhibitor bendiocarbamate. According to literature, bendiocarbamate has relatively low toxicity in mammals and vertebrates in general, since it does accumulate in their tissues and the cholinesterase activity returns to norm within 24 h after acute exposure. The present study focused on the influence of bendiocarbamate on rabbit thymus after its administration at a dose of 5 mg/kg body weight (BW) for the period of 3 months. The thymus was observed for acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE)-positive nerve fibers visualized by histochemical methods. Microscopic findings of BuChE-positive nerve fibers show the same density and the topography by the experimental and the control animals. On the other hand, AChE-positive nerve fibers in experimental animals after administration of bendiocarbamate is only poorly identified, suggesting that bendiocarbamate inhibits AChE but not BuChE.     

Fish Cholinesterases as Biomarkers of Sublethal Effects of Organophosphorus and Carbamates in Tissues of Labeo Rohita

Organophosphates and carbamates are major agrochemicals that strongly affect different neuroenzymes and the growth of various fish species. Here, we study the effect of sublethal concentrations of profenofos and carbofuran on the activity of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) and the associated health risk in fish. Labeo rohita fingerlings were exposed to three sublethal concentrations of profenofos and carbofuran. The minimum cholinesterase activities in the brain, gills, muscle, kidney, liver, and blood were after exposure to profenofos (0.06 mg/L). The minimum AChE and BuChE activities in the brain, gills, muscle, kidney, liver, and blood were after exposure to carbofuran (0.28 and 0.198 mg/L). Exposure to both types of pesticides affected the functions of these organs, including metabolism and neurotransmission, to various extents at different exposure concentrations. These findings suggest that they are required to be properly monitored in the environment, to reduce their toxic effects on nontarget organisms     

Human butyrylcholinesterase components differ in aryl acylamidase activity

Apart from its esterase activity, butyrylcholinesterase (BuChE) displays aryl acylamidase (AAA) activity able to hydrolyze o-nitroacetanilide (ONA) and its trifluoro-derivative (F-ONA). We report here that, despite amidase and esterase sites residing in the same protein, in human samples depleted of acetylcholinesterase the ratio of amidase to esterase activity varied depending on the source of BuChE. The much faster degradation of ONA and F-ONA by BuChE monomers (G1) of colon and kidney than by the tetramers (G4) suggests aggregation-driven differences in the AAA site between single and polymerized subunits. The similar ratio of F-ONAto butyrylthiocholine hydrolysis by serum G1 and G4 forms support structural differences in the amidase site according to the source of BuChE. The changing ratios of amidase to esterase activities in the human sources probably arise from post-translational modifications in BuChE subunits, the specific proportion of monomers and oligomers and the variable capacity of the tetramers for degrading ONA and F-ONA. The elevated amidase activity of BuChE monomers and the scant activity of the tetramers justify the occurrence of single BuChE subunits in cells as a means to sustain the AAA activity of BuChE which otherwise could be lost by tetramerization.     

Cholinesterase in muscle of dystrophic hamsters (Bio-40.54)

Isozyme patterns of cholinesterase (ChE) from heart, tongue, and skeletal muscle of normal and dystrophic hamsters are presented. Two principal bands, bands 1 and 2, were evaluated. Band 1 migrates faster towards the anode than does band 2. While bands 1 and 2 stain for AChE and were found in control muscles, only band 2 was stained by a pseudocholinesterase (BuChE) and was decreased in samples from dystrophic hamsters. The decrease in BuChE was most pronounced in dystrophic heart muscle. The low level of BuChE measured for dystrophic animal tissue was similar to isozyme patterns found in embryonic tissue and in denervated muscle. BuChE obtained by acrylamide gel electrophoresis along with 16S AchE appears to be a useful biochemical marker of nerve-muscle interactions.     

Charaterization and hydrodynamic behaviour of modified gelatin: II. Characterization by high performance size exclusion chromatography — comparison with dextrans and proteins

Gelatin samples obtained by chemical modification (succinylation) are studied by SEC on silica based chromatographic supports. The influence of the pH of eluent mixtures (potassium phosphate added to NaCl) in the range 7-3.3 shows that the void volume peak (VVP) is lowered or even vanishes at pH 3.3 with the 3000 SW (TSK) gel. A process using an ultrasound treatment before injection is reported in order to determine accurately the molecular parameters of gelatin onto TSK gel with a minimal VV P. This peak is attributed to molecular aggregation of a part of the modified gelatin. After disaggregation by ultrasound or heat treatment the results are in good accordance with those obtained by other methods. It is demonstrated that with proteins and dextrans the TSK 3000 SW gel does not agree with the universal calibration curve (log[ν] · versus K_d as reported previously. A single calibration curve is obtained when the Stokes radius is plotted versus K_d. Gelatin fractions are eluted at pH 7 close to this calibration curve. This plot shows that gelatin fractions at pH 3.3 are not eluted by a pure size exclusion mechanism on 3000 SW gel. It is concluded that hydrophobic interactions between fractions of gelatin and the gel explain the high retention of these samples.     

Effect of pesticide exposure on the cholinesterase activity of the occupationally exposed tea garden workers of northern part of West Bengal,India

Context: Pesticide poisoning and related deaths are a global concern, but there is little information about its effect on the occupationally exposed tea garden workers of North Bengal.

Objective: This study investigates the level of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in the blood of the tea garden workers at risk of exposure to a mixture of pesticides.

Materials and methods: The study sample consisted of pesticide exposed workers, non-exposed (control), smokers and alcoholics. AChE and BuChE activity was measured and tested for significance.

Results: Results showed that AChE activity was half in the pesticide exposed individuals than controls (p≤ 0.001). BuChE activity was also significantly decreased in the pesticide exposed individuals than controls (p≤ 0.001), while AChE and BuChE activity in smokers and alcoholics were not different from that of controls. However, significantly decreased AChE and BuChE activities were recorded in pesticide exposed workers compared to smokers and alcoholics.

Conclusions: The results indicated that the decrease in enzyme activities in tea garden workers was due to mixed pesticides (containing organophosphates) exposure. Age was not found to influence the enzyme activities. However, the gender had little effect on the enzyme activities but the effect was not so prominent.     

Pepsinogen B: the zymogen of pepsin B

1. Pepsinogen B, the precursor of pepsin B, has been isolated by ion-exchange chromatography and gel filtration from neutral extracts of pig gastric mucosa. The material possesses potential activity against acetyl-l-phenylalanyl-l-di-iodotyrosine and against gelatin but has little, if any, potential activity against haemoglobin. 2. The material appears homogeneous in the ultracentrifuge, but on gel filtration and on electrophoresis in starch gel it is shown to be contaminated with a small amount of material having potential activity against haemoglobin. On electrophoresis in starch gel also the material is shown to contain about equal amounts of two major components, both of which have potential activity against the synthetic substrate. Pepsin B has also been shown to contain two active components by electrophoresis under the same conditions. 3. The zymogen is similar to pepsinogen and pepsinogen C in its molecular weight and general physico-chemical properties, but differs from these zymogens in the nature of its N-terminal residues. It is possible that one of the components contains 1 mole of bound phosphate/mole. 4. The material is activated rapidly at pH2 and more slowly at pH4. At both pH values the kinetics of the activation reaction are complex.     

Inter-tissue and inter-species comparison of butyrylcholinesterases

Butyrylcholinesterase (BuChE) occurs in a multiple molecular forms whose catalytic activity depends on tissue distribution and species. The hypothesis led us to the study of BuChE catalytic properties focused on the inter-tissue and inter-species level with benzoylcholine and N-alkyl derivates of benzoylcholine (BCHn) as substrates. These compounds are soft disinfectants easily biodegradable to biologically inactive hydrolytic products, substituted choline and benzoic acid. Different sources of BuChE were used: rabbit and rat liver microsomal fraction (membrane-anchored enzyme) and serum (soluble form). Hydrolytic activity of both these BuChE forms was compared to rat recombinant BuChE (rBuChE). Hydrolytic product (benzoic acid) formation was recorded as function of time, and hydrolytic rate was determined. Tissue distribution of BuChE plays an important role in hydrolysis of BCHn. High BuChE activity was observed in a serum of both studied species rat and rabbit and was significantly dependent on a structure of substrates. Activity of soluble serum forms was the same as that for the rBuChE. Significant change of BuChE activity was recorded on the inter-species level in the microsomal fractions. It is because the rabbit microsomal BuChE activity had absolutely different course for all substrates as compared to rat microsomes. Inhibitory studies of BCHn enzymatic hydrolysis of all BuChE forms were performed to determine the level of BuChE participation in BCHn hydrolysis. It can be concluded that short-chain BCHn substrates are exclusively hydrolyzed by BuChE from all studied sources except for the rabbit liver microsomal fraction. Rabbit seems to have different enzymes involved in the hydrolysis of all studied BCHn compounds.     

Localization and Properties of Enzymes Involved with Electron Transport Activity in Mycobacteria

A study of the subcellular localization of the nicotinamide adenine dinucleotide (NADH)-3-(4, 3-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) oxido-reductase systems in Mycobacterium was presented. Evidence based on starch gel electrophoresis and different responses of the subcellular fractions to heat inactivation suggested the existence of more than one enzyme responsible for the NADH-MTT oxido-reductase activity. One type of activity was found in the membrane-mesosome fraction which contained labile and electrophoretically non-migrating enzymes. Another type of activity was also detected in the soluble fraction which on starch gel electrophoresis exhibited 4 bands of activity, two of which showed heat resistance.     

Temperature—composition phase diagram and gel properties of the gelatin—starch—water system

The gelatin-starch-water system has been studied at different temperatures, at a total biopolymer concentration of 5.0 wt%. The weight ratios (W) of gelatin/ starch used were 9:1, 8:2.4. 2:8, 1:9, with pH values between 5.82 (at W = 9:1) and 6.50 (at W = 1:9). The systems were characterized rheologically and by turbidity measurements to construct a phase diagram in the temperature (T) and composition (W) variables. The T-W quadrant consists of three regions: a singlephase solutions region (A) and regions of complete and incomplete phase separation (B and C, respectively). The system in region C is a gel. Region B, lying between A and C, corresponds to two co-existing liquid phases. The transition from A to C (obtained by cooling the system at constant W) involves crossing region B. The properties of the resulting gels depend on the rate of this intersection. Gels formed on rapid cooling have an even distribution of turbidity, whereas slow cooling gives two gel layers of different turbidity. The gelation temperature and gel strength of the mixed systems are dominated by the gelatin component, with no indication of network formation by starch.     

Species variation in the distribution of cholinesterases in the ovary of the plains viscacha, cat, ferret, rabbit, rat, guinea-pig and roe deer

Synopsis Sections of ovary from plains viscacha, cat, ferret, rabbit, rat, guinea-pig and roe deer have been histochemically processed to demonstrate acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in nervous and non-nervous tissue. The effects of different reproductive states on enzyme activity were observed in some animals. AChE-containing nerves were sparse in rabbit and rat but plentiful in cat and roe deer. Nerves containing BuChE were not detectable in ferret or guinea-pig and were rare in cat. Species variations in the activity and type of enzyme were also found in non-neuronal tissues. Some blood vessels in the ovaries of guinea-pig and viscacha contained AChE. No other species showed a reaction for AChE in non-neuronal stromal tissue but BuChE was present at this site in all animals except rat. Granulosa cells reacted for AChE only in cat and rabbit while luteal cells were reactive in cat, rabbit and roe deer. Some BuChE activity was present in granulosa and or luteal cells in all species except roe deer. In rat, BuChE activity in luteal cells increased during oregnaney and the early phase of pseudopregnancy. The difficulty of assigning a function to ovarian cholinesterases is discussed.     

Butyrylcholinesterase-Mediated enhancement of the enzymatic activity of trypsin

1. Acetylcholinesterase (AChE, EC 3.1.1.7) and butyrylcholinesterase (BuChE, EC 3.1.1.8) are enzymes that catalyze the hydrolysis of esters of choline.2. Both AChE and BuChE have been shown to copurify with peptidases.3. BuChE has also been shown to copurify with other proteins such as transferrin, with which it forms a stable complex. In addition, BuChE is found in association with -amyloid protein in Alzheimer brain tissues.4. Since BuChE copurifies with peptidases, we hypothesized that BuChE interacts with these enzymes and that this association had an influence on their catalytic activities. One of the peptidases that copurifies with cholinesterases has specificity similar to trypsin, hence, this enzyme was used as a model to test this hypothesis.5. Purified BuChE causes a concentration-dependent enhancement of the catalytic activity of trypsin while trypsin does not influence the catalytic activity of BuChE.6. We suggest that, in addition to its esterase activity, BuChE may assume a regulatory role by interacting with other proteins.     

Detection of human butyrylcholinesterase-nerve gas adducts by liquid chromatography-mass spectrometric analysis after in gel chymotryptic digestion

To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis.     

Butyrylcholinesterase fluctuation in male albino rats with intracerebroventricular injection of gamma aminobutyric acid, muscimol, and picrotoxin

The effects of intracerebroventricular injection of gamma-Aminobutyric acid, muscimol, or picrotoxin have been studied on butyrylcholinesterase (BuChE) activities in the serum and several hypothalamic nuclei using biochemical, histochemical, and cytophotometric techniques, respectively. The blood samples were withdrawn from indwelling catheters in jugular vein 1, 15, 30, 45, 60, 90, and 120 min after injection of the drugs. Biochemical estimations demonstrated a significant inhibition of BuChE after GABA and muscimol injections, whereas a pronounced stimulation of BuChE was observed after injection of picrotoxin. The peak changes were observed within 30 min of drug injection. Cytophotometric studies have appeared to dovetail the biochemical findings. Only a marginal decrease was observed after injection of GABA in all nuclei, while muscimol induced a very conspicuous decrease of BuChE. On the contrary, intracerebroventricularly administered picrotoxin markedly increased the levels of BuChE activity. Thus it could be concluded that probably GABA and muscimol along with picrotoxin appear to alter BuChE.     

Differential effect of pressure and temperature on the catalytic behaviour of wild-type human butyrylcholinesterase and its D70G mutant.

The combined action of temperature (10-35 degrees C) and pressure (0. 001-2 kbar) on the catalytic activity of wild-type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild-type BuChE (at Tt approximately 22 degrees C) whatever the pressure (dTt/dP = 1.6 +/- 1.5 degrees C.kbar-1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature-induced conformational change of the wild-type BuChE which did not occur for the D70G mutant. For the wild-type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild-type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 degrees C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.     

Enzymes of starch metabolism in the developing rice grain

The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and starch synthetase —were assayed in dehulled developing rice grains (Oryzasativa L., variety IR8). Phosphorylase, Q-enzyme, and R-enzyme had peak activities 10 days after flowering, whereas α- and β-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule.     

Butyrylcholinesterase activity and molecular components in thymus of healthy and merosin-deficient Lama2dy mice

The laminin-alpha2 chain, referred to as merosin, forms part of the laminin-2 heterotrimer (alpha2beta1gamma1), which is principally expressed in the basement membrane of muscle. Nearly half of patients suffering from congenital muscular dystrophy (CMD) have abnormalities in the laminin-alpha2 chain (LAMA2) gene, and the merosin-deficient Lama2dy mouse shows CMD. The expression of merosin in thymus, the abnormalities in the gland of Lama2dy mice, and the presence of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) in thymus prompted us to study the possible effects of the deficiency of merosin on thymus BuChE. We found that, while AChE activity decreased by approximately 50% in merosin-deficient thymus, the deficiency had little effect on BuChE activity. About 65% of thymus BuChE activity was extracted with a saline buffer and 30% with 1% Triton X-100. Sedimentation analyses and phenyl-agarose chromatography showed that thymus contained amphiphilic BuChE monomers (G(1)(A),44%) and dimers (G(2)(A),33%), and hydrophilic tetramers (G(4)(H),23%). Binding assays with various plant lectins revealed differences between the oligoglycans linked to BuChE tetramers and lighter components. The deficiency of merosin had no effect on the biosynthesis of thymus BuChE as judged by the lack of major changes between control and Lama2dy mice thymuses in the distribution of BuChE molecules and the level of lectin binding. The detoxifying action of BuChE, its role as a backup to AChE, and the relevance of the cholinergic dialogue between T cells and stromal cells for T lymphocyte proliferation, maturation and survival support a physiological function for BuChE in thymus.