Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Butyrylthiocholine”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1999

Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants.

Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) variants was measured in phosphate buffer pH 7.4 at 25 degrees C. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where there was a small decrease. To obtain the catalytic constants, three equations were applied: Michaelis-Menten equation (Eq. 1), Hill equation (Eq. 2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (Eq. 3). Over a range from 0.01 to 50 mM BTCh, the activity versus substrate concentration relationship deviated from Michaelis-Menten kinetics (Eq. 1) while data fitted well with Eqs. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges: the corresponding Km constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01-1.0 mM BTCh) and from 0.3 to 2.0 mM (at 1.0-50 mM BTCh). Hill coefficients (nH) calculated from Eq. 2 were similar for all phenotypes (nH approximately 0.5). The dissociation constants K1 and K2 calculated from Eq. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K1) and 0.89 and 4.9 mM (K2) for the five phenotypes. Experimental data support the assumption that the phenotypes studied have two substrate binding sites.

Binding Sites↗
PubMed1997

Mechanism of eserine action on the hydrolysis of butyrylthiocholine by butyrylcholinesterase.

The mechanism of the interaction of eserine with butyrylcholinesterase has been proposed only on the basis of analogy with acetylcholinesterase. Here the interactions was studied in detail and the results analysed by classical kinetic methods and by means of mathematical modelling. An appropriate kinetic scheme was developed, an adequate equation derived and the corresponding kinetic parameters evaluated. The findings suggest that a fast but relatively weak binding of eserine to the enzyme's active site is followed by a slow acylation step and by an even slower rate limiting deacylation step so misrepresenting eserine as an irreversible inhibitor. The proposed kinetic scheme also suggests that the reaction of eserine with a peripheral substrate site is unlikely as seen with the substrate, butyrylthiocholine.

Binding, Competitive↗
PubMed1983

[Cholinesterase (EC 3.1.1.8) with butyrylthiocholine-iodide as substrate: references depending on age and sex with special reference to hormonal effects and pregnancy].

Referance values are reported for the assay of cholinesterase (substratae: butyrylthiocholine iodide) in serum at 25 degrees C. There was no evidence of any age-dependency or sex-specific distinction in children (1 to 15 years, N = 309), nor were there any age-dependent changes observed in males (16 to 94 years, N = 718). Compared with the reference range for children, there was no detectable difference in the location and distribution of catalytic cholinesterase concentrations. In females (16 to 99 years, N = 861), an age-dependent difference in cholinesterase values became apparent: Regardless of pregnancy or use of hormonal contraceptives, the catalytic concentrations were found to be lower in younger females (16 to 39 years) than in older ones (greater than or equal to 40 years); the reference values in the older age group (40 to 99 years) did not differ from those of males and children. Therefore the following reference values are proposed (in each case 2.5 to 97.5 percentile: Children, males and females above 40 Years: 3.5 to 8.5 kU/l, Females (16-39 years, nonpregnant, not taking hormonal contraceptives): 2.8 to 7.4 kU/l, Females (18-41 years, pregnant or taking hormonal contraceptives): 2.4 to 6.0 kU/l.

Adolescent↗
PubMed2003

High activity of human butyrylcholinesterase at low pH in the presence of excess butyrylthiocholine.

Butyrylcholinesterase is a serine esterase, closely related to acetylcholinesterase. Both enzymes employ a catalytic triad mechanism for catalysis, similar to that used by serine proteases such as alpha-chymotrypsin. Enzymes of this type are generally considered to be inactive at pH values below 5, because the histidine member of the catalytic triad becomes protonated. We have found that butyrylcholinesterase retains activity at pH <or= 5, under conditions of excess substrate activation. This low-pH activity appears with wild-type butyrylcholinesterase as well as with all mutants we examined: A328G, A328I, A328F, A328Y, A328W, E197Q, L286W, V288W and Y332A (residue A328 is at the bottom of the active-site gorge, near the pi-cation-binding site; E197 is next to the active-site serine S198; L286 and V288 form the acyl-binding pocket; and Y332 is a component of the peripheral anionic site). For example, the kcat value at pH 5.0 for activity in the presence of excess substrate was 32900 +/- 4400 min(-1) for wild-type, 55200 +/- 1600 min(-1) for A328F, and 28 700 +/- 700 min(-1) for A328W. This activity is titratable, with pKa values of 6.0-6.6, suggesting that the catalytic histidine is protonated at pH 5. The existence of activity when the catalytic histidine is protonated indicates that the catalytic-triad mechanism of butyrylcholinesterase does not operate for catalysis at low pH. The mechanism explaining the catalytic behaviour of butyrylcholinesterase at low pH in the presence of excess substrate remains to be elucidated.

Amino Acid Substitution↗
PubMed1988

Cholinesterases in rabbit serum.

1. Rabbit serum was shown to contain two cholinesterases which hydrolysed acetylthiocholine and butyrylthiocholine and one cholinesterase which hydrolysed only butyrylthiocholine. 2. The three enzymes were identified by the kinetics of heat inactivation and kinetics of phosphorylation by the organophosphate VX. 3. Using selective inhibitors (iso-OMPA, eserine, BNPP and BW-284C51) it was shown that the hydrolysis of acetylthiocholine and butyrylthiocholine in untreated native serum had properties of acetylcholinesterase (EC 3.1.1.7), butyrylcholinesterase (EC 3.1.1.8) and also some properties of carboxylesterase (EC 3.1.1.1). 4. Separation of proteins (on PAA-gels) in untreated native serum gave four bands with acetylthiocholine and three with butyrylthiocholine. 5. The two cholinesterases hydrolysing both substrates corresponded to the slow moving bands on the gel. 6. The fastest moving band hydrolysing only butyrylthiocholine could be attributed to the cholinesterase least sensitive to VX.

Acetylthiocholine↗
PubMed2001

Determination of whole blood cholinesterase in different animal species using specific substrates.

Whole blood cholinesterase was measured using acetyl-, butyryl- and propionylthiocholine as substrates in 10 healthy adult dogs, cats, horses, pigs, goats, sheep and cows, in order to determine and characterise the cholinesterase activity in whole blood of the main domestic animals. An in vitro exposure test with two anticholinesterase compounds, the organophosphate insecticide coumaphos and the carbamate insecticide imidocarb, was also performed. In whole blood of ruminants and pigs, acetylthiocholine yielded the highest cholinesterase activity and other substrates were poorly hydrolysed; in dogs and cats, although acetylthiocholine showed the highest cholinesterase activity, butyryl- and propionylthiocholine also produced high cholinesterase values; in horses, propionylthiocholine was the substrate that yielded the highest cholinesterase activity, closely followed by butyrylthiocholine. All within- and between-run coefficients of variation observed in whole blood samples were less than 5 and 7 per cent, respectively, except when butyrylthiocholine was used as substrate in ruminant blood samples. Butyryl- and propionylthiocholine were the substrates that yielded higher inhibitions after coumaphos exposure, whereas the use of acetylthiocholine showed the highest cholinesterase inhibition after imidocarb exposure. The use of at least two substrates (acetyl and butyrylthiocholine) is recommended for whole blood cholinesterase analyses in domestic animals since it will allow monitoring of both acetyl- and butyrylcholinesterase activities, respectively, and a more accurate detection of exposure to anticholinesterase compounds. However, acetylthiocholine could be used as a unique substrate for whole blood cholinesterase determination in porcine and ruminant samples since butyrylcholinesterase activity is very low in these species. Additionally, propionylthiocholine could be used as an alternative substrate to butyrylthiocholine in horse whole blood samples.

2,2'-Dipyridyl↗
PubMed1984

Substrate-dependent kinetic behavior of horse plasma cholinesterase: evidence for kinetically distinct populations of active sites.

The inhibition of horse plasma cholinesterase by propranolol showed characteristics which depended upon the identity of the substrate used. With butyrylthiocholine as substrate, the inhibition showed a first-order dependence on inhibitor concentration, and was characterized by a Ki of 8 microM (pH 7.4, 20 degrees C). With p-nitrophenylbutyrate as substrate, a biphasic v-1 versus [I] relationship was obtained. The biphasic curve could be resolved into two components, with apparent Ki's of 9 microM and 1.3 mM. Use of butyrylthiocholine as alternative substrate resulted in partial inhibition of p-nitrophenylbutyrate hydrolysis. Inhibition of butyrylthiocholine hydrolysis by p-nitrophenylbutyrate could be accounted for by pure competitive inhibition at two sites. The results were interpreted in terms of a four-site, low-symmetry model, in which two active sites could process both substrates, and the remaining sites could process only p-nitrophenylbutyrate.

Allosteric Site↗
PubMed2000

Use of whole blood for spectrophotometric determination of cholinesterase activity in dogs.

Whole blood has been compared with erythrocytes and plasma for spectrophotometric cholinesterase determination in the dog. Cholinesterase activity was characterized using two substrates: acetylthiocholine and butyrylthiocholine. Acetylcholinesterase was the only form of cholinesterase present on erythrocytes and hydrolysed only acetylthiocholine. Butyrylcholinesterase (pseudocholinesterase) was predominant in plasma, hydrolysing mainly butyrylthiocholine. Based on these results, a method based on the use of two substrates (acetylthiocholine for monitoring acetylcholinesterase and butyrylthiocholine for determining butyrylcholinesterase) in the same whole blood sample is recommended for canine cholinesterase analysis. This way of monitoring both enzymes can be easily automated, yielding good within (CVs < 5%) and between-run (CVs < 7%) precision.

2,2'-Dipyridyl↗
PubMed1996

Asp7O in the peripheral anionic site of human butyrylcholinesterase.

The goal of this work was to determine what amino acids at the mouth of the active-site gorge are important for the function of human butyrylcholinesterase. Mutants D70G, Q119Y, G283D, A277W, A277H and A277W/G283D were expressed in human embryonal kidney cells and the secreted enzymes were assayed by steady-state kinetics. The result was that only one amino acid, D70 was found to be important for function. When D70 was mutated to G, the same mutation as in the naturally occurring atypical butyrylcholinesterase, the affinity for positively charged substrates and positively charged inhibitors decreased 5-30-fold. The D70G mutant had another striking abnormality in that it was virtually devoid of the phenomenon of substrate activation by excess butyrylthiocholine. Thus, though kcat was the same for wild-type and D70G mutant, being 24000 min(-1) at low butyrylthiocholine concentrations (0.01-0.1 mM), it failed to increase for the D70G mutant at 40 mM butyrylthiocholine, whereas it increased threefold for wild type. The D70G mutant was more sensitive to changes in salt concentration, its catalytic rate decreasing more than that of the wild type. The D70G mutant appeared to have a greater surface negative charge than wild type suggesting that the D70G mutant had a conformation different from that of the wild type. That D70 affects the function of butyrylcholinesterase, together with its location at the mouth of the active-site gorge, supports the hypothesis that D70 is a component of the peripheral anionic site of butyrylcholinesterase. Mutants containing aromatic amino acids at the mouth of the gorge had increased binding affinity for propidium and fasciculin, but unaltered function, suggesting that aromatic amino acids are not important to the function of the peripheral anionic site of butyrylcholinesterase.

Acetylcholinesterase↗
PubMed1983

Comparison of a commercially available assay system with two reference methods for the determination of plasma cholinesterase variants.

For assaying plasma cholinesterase (EC 3.1.1.8) activity and phenotyping by means of dibucaine inhibition, we have compared a commercially available kit, in which butyrylthiocholine is used as substrate, with two reference methods, one using benzoylcholine and the other propionylthiocholine. With 50 different samples of three of the most common genetic variants, we could clearly differentiate the variants with benzoylcholine and dibucaine, whereas there was some overlap of the E1uE1u and E1uE1a phenotypes with the other two substrates at 30 degrees C. The phenotypes were better differentiated at 25 degrees C, and in our hands the use of butyrylthiocholine was preferable to propionylthiocholine for phenotyping with dibucaine. The affinity of the usual and atypical homozygotes for fluoride with butyrylthiocholine gave an inverted response to the affinity of these variants for the anion with benzoylcholine. We suggest that this may be explained by the role of the chromogen or its products in the assay procedure with the thiocholine substrate.

Benzoylcholine↗
PubMed1993

Improved colorimetric method for cholinesterase activity.

A modified colorimetric method for the estimation of cholinesterase activity has been worked out using two different substrates, acetylthiocholine iodide for total cholinesterase and a specific substrate, butyrylthiocholine iodide for pseudocholinesterase in the same sample. This is a modification of the method described by Voss and Sachsse (1970) wherein acetylthiocholine iodide was used for both total and pseudo cholinesterase activities. The pseudocholinesterase obtained with acetylthiocholine iodide was significantly higher (P < 0.0001) than that with butyrylthiocholine iodide either in whole blood or serum samples. Acetylthiocholine iodide while reacting with pseudocholinesterase in serum or plasma samples might also be interacting with the small quantities of acetylcholinesterase present. It is therefore suggested that butyrylthiocholine iodide and acetylthiocholine iodide may be used to determine pseudocholinesterase and total cholinesterase activities respectively. The use of two substrates with a few more alterations in the experimental conditions increased the validity of this simple and rapid colorimetric method.

Acetylthiocholine↗
PubMed1987

Differential reactivity of active sites in human plasma cholinesterase toward 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.

Human plasma cholinesterase was found to be inhibited by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a biphasic manner. The faster phase of the inhibition led to loss of approximately 50% of the activity (measured at pH 7.0, 30 degrees C, using 2.5 mM butyrylthiocholine) and was irreversible. Inhibition in the slower phase was reversible by 0.25 M hydroxylamine. The protective effect of 1 mM propranolol indicated that the target residue in both phases was localized at the active site. Lineweaver-Burk plots for butyrylthiocholine were obtained at different times during the course of inactivation. It was found that for both native and partially inactivated enzymes the plots could be analyzed in terms of two activities showing hyperbolic saturation with the substrate, with Km values of 0.055 +/- 0.015 and 2.0 +/- 0.2 mM. The carbodiimide affected the maximal velocities of the component activities, leaving the Km's unchanged. The low-Km component was lost in the first phase of the inactivation. The loss of the high-Km component paralleled the second phase. It was concluded that the active sites in the tetrameric enzyme form two classes, differing in their affinity for butyrylthiocholine and their susceptibility to inhibition by the active site-directed carbodiimide.

Binding Sites↗
PubMed1995

Catalytic properties of rabbit serum esterases hydrolyzing esterified monosaccharides.

Rabbit serum and one enzyme fraction isolated from rabbit serum by column chromatography (Fraction II) were used as catalysts in regioselective hydrolysis of radiolabelled pivaloylated monosaccharides (Piv = Me3CCO). The hydrolysis of 14C-labelled methyl 2-O-pivaloyl-(2-MP)-, 6-O-pivaloyl (6-MP)-, 2,6-di-O-pivaloyl-(2,6-DP) alpha-D- glucopyranosides and methyl 2-acetamido-2-deoxy-3,6- di-O-pivaloyl-(3,6-DPNAc) alpha-D-glucopyranosides, was studied, as well as that of the non-sugar substrates butyrylthiocholine, thiophenylbutyrate, phenylacetate and paraoxon. The specific activities of 2,6-DP, 3,6-DPNAc, butyrylthiocholine and thiophenylbutyrate were higher in Fraction II than in native sera, while those of phenylacetate and paraoxon were lower. Inhibition studies were done using the substrates mentioned and five different inhibitors, namely bis(p-nitrophenyl phosphate) (BNPP), eserine, paraoxon, HgCl2 and EDTA. The hydrolysis of 2,6-DP and 3,6-DPNAc was not inhibited by HgCl2 and only slightly by EDTA. Paraoxon, eserine and BNPP were progressive inhibitors of the hydrolysis of the two sugar substrates, and the pattern of inhibition resembled closely the inhibition of butyrylthiocholine and thiophenylbutyrate hydrolysis. This result applied to both, native serum and Fraction II. It was concluded that esterases in rabbit serum which hydrolyze pivaloylated sugar substrates belong to the category of serine esterases. Kinetic parameters (KM and Vmax), effects of temperature and pH on activity of esterases from Fraction II were also determined for the hydrolysis of sugar substrates.

Animals↗