Human Plasma Butyrylcholinesterase Research Articles

A stability index for enzyme deactivations

An empirical stability index is proposed for single-step and two-step series-type deactivations. The stability index makes more quantitative the effect of different variables or modification on enzyme stability. The stability index increases linearly with reciprocal temperature for soluble alcohol dehydrogenase, α-amylase, human plasma butyrylcholinesterase, and human plasma butyrylcholinesterase in deuterium oxide. Other examples of enzyme modifications and their effect on the stability index are also given.

Stability of butyrylcholinesterase: thermal inactivation in water and deuterium oxide.

Irreversible thermal inactivation of the tetrameric form of human plasma butyrylcholinesterase (cholinesterase; EC 3.1.1.8) was studied in water and in deuterium oxide at pH 7 in the temperature range 53-65 degrees C. The enzyme inactivation follows a complex kinetics that may be described by the sum of two apparent first-order processes. The Eyring plot for enzyme inactivation exhibits a wavelike discontinuity over a span of 2 C degrees around 58 degrees C. This transition was interpreted in terms of equilibrium between two temperature-dependent conformational states. Though 2H2O does not alter the overall multistep inactivation process, a slight solvent isotope effect was observed: a stabilizing effect and a shift in the transition temperature. A comparison between several enzyme preparations revealed differences in thermodynamic activation parameters of inactivation suggesting microheterogeneity in enzyme structures. Kinetics of inactivation of usual (E1uE1u) and atypical (E1aEa1a++) enzymes were compared. The atypical enzyme was found to be more stable than the usual phenotype.

Structure-activity relationship of reversible cholinesterase inhibitors including paraquat.

The inhibitory effect of paraquat on cholinesterase activity was investigated in comparison with four paraquat derivatives, six monoquaternary ammoniums and six anticholinergic drugs. Inhibitor concentrations to cause 50% inhibition (I50) and Hill coefficients for three enzymes, human erythrocyte acetylcholinesterase (AChE), Electrophorus electricus AChE and human plasma butyrylcholinesterase (BuChE) were measured. The results obtained were as follows. The I50 for erythrocyte AChE was similar to the I50 for eel AChE. Secondary to edrophonium, diethylparaquat, paraquat, morfamquat and monoquat showed lower I50 for AChE, and possessed higher inhibition selectivity (IS), expressed as the ratio of I50 for BuChE to I50 for erythrocyte AChE. However, diquat showed higher I50 for AChE and lower IS, similar to the other monoquaternary ammoniums. A negative correlation was observed between log [I50 for erythrocyte AChE] and log [IS], among paraquat and its derivatives, monoquaternary ammoniums and anticholinergic drugs, respectively. With respect to Hill coefficients, these inhibitors could be classified into four groups, [1] competitive inhibitors: diquat, edrophonium, choline, tetramethylammonium and trimethylphenylammonium, [2] inhibitors showing negative cooperativity: paraquat, diethylparaquat, morfamquat, d-tubocurarine, atropine, gallamine and nicotine, [3] moderate type inhibitors: monoquat, hexamethonium and decamethonium. [4] the other type inhibitors showing positive cooperativity for erythrocyte AChE: tetraethylammonium and ethyltrimethylammonium.

Monoclonal antibodies to human brain acetylcholinesterase: properties and applications.

1. Acetylcholinesterase (AChE) was purified 20,000-fold in a 43% yield from 90 g of human cerebellum by combined immunoaffinity and ligand affinity chromatography. The purified enzyme migrated as a 68,000-dalton band during polyacrylamide gel electrophoresis under denaturing and reducing conditions. 2. Balb/c mice were immunized with multiple 10-micrograms injections of this material in order to raise monoclonal antibodies to human brain AChE. Three such antibodies were obtained and characterized. 3. Each antibody cross-reacted distinctively with AChEs from other mammals. No antibody recognized human plasma butyrylcholinesterase but all reacted with AChE from human red blood cells. 4. Antibodies HR5 and HR3 performed well in two-site immunoassays for AChE. With these assays we compared autopsy samples of cortical region A9 from six controls (nonneurological cases) and five patients with Alzheimer's disease. The latter showed a highly significant 60% deficit of AChE protein. 5. The present antibodies will permit additional immunochemical studies of cholinergic systems in dementia.

Molecular forms of acetylcholinesterase and butyrylcholinesterase in human plasma and cerebrospinal fluid.

The measurement of cholinesterase activities in either plasma or cerebrospinal fluid (CSF) may ultimately prove to be relevant in the diagnosis of neurological and neuropsychiatric disorders. However, studies to date have examined only total enzyme activities. Therefore in the present study we have examined the distribution of the individual molecular forms of both acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) in plasma and CSF using sucrose density gradient centrifugation. Although the total activities of AChE were of the same order of magnitude in plasma and CSF, there was a considerable difference (120-500-fold) between total BChE activity in the CSF and the BChE-rich plasma. The analysis of the individual molecular forms revealed that the predominant molecular species of AChE and BChE in the CSF--both lumbar and ventricular--was the G4 form. The G4 form also constituted the majority of the plasma BChE activity and, on average, over half (56%) of the plasma AChE activity. The significance of the AChE and BChE molecular form compositions of both plasma and CSF and their possible relationship to pathological states are discussed.

Horizontal two-dimensional electrophoresis of plasma butyrylcholinesterase.

Genetic variants of human plasma butyrylcholinesterase have been characterized and are highly relevant to anesthesiology. They might also represent potential genetic markers for neuropsychiatric disorders. Two-dimensional electrophoresis with isoelectrofocusing in the first and polyacrylamide gel electrophoresis in the second dimension has proved to be a powerful tool in search for genetic variants. Butyrylcholinesterase is an oligomeric enzyme with considerable charge heterogeneity. Conventional two-dimensional electrophoresis proved unsuitable for this enzyme possibly due to its tendency to aggregate by hydrophobic interactions. The inversion of the sequence applying polyacrylamide gel electrophoresis in the first and isoelectric focusing in the second dimension circumvented this problem.

Etude électrophorétique de la butyrylcholinestérase agée après inhibition par le soman

The following states of purified tetrameric form (C 4) of human plasma butyrylcholinesterase were studied by electrophoretic techniques: native, inhibited by soman and by methane sulfonyl fluoride and soman-aged. In order to detect a significant conformational change of the aged cholinesterase as compared to the non-inhibited (native) species, enzymes were treated with a set of bifunctional reagents (diimidates) of different chain lengths. After denaturation, the cross-link products were subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis. The peak areas of the cross-linked species and the parameters of cross-linkability were calculated from densitometric data, versus the maximal effective reagent length. The effect of occupancy of the esteratic site by substituted phosphonyl group and by methyl-sulfonyl residue on the binding activity of the anionic site was studied by affinity electrophoresis at varying temperatures with immobilized-procaïnamide as ligand. Apparent dissociation-constants of the enzyme-ligand complexes were estimated from measurement of mobilities versus ligand concentration. Corresponding thermodynamic quantities were calculated from Van't Hoff plots and basic thermodynamic equations. The reactivity of aged-cholinesterase with diimidates was similar to that of the native enzyme. Affinity for immobilized-procaïnamide was slightly lowered in aged and inhibited enzymes as compared to the native and sulfonylated enzymes. As for the ligand-induced isomerization of anionic site (A → B), revealed by affinity electrophoresis, the ligand concentration at the midpoint of transition ( A = 0.5 ) was slightly greater for the aged enzyme than for the native one. From these results, the following conclusions can be drawn: the dealkylation of somancholinesterase conjugate (aging) does not seem to induce structural changes detectable in the cross-linkability of lysyle residues at the subunit interfaces andthe surface of the tetrameric enzyme. On the other hand, the affinity of the anionic site and ligand-induced isomerization process are altered in soman-inhibited and aged enzymes. These data suggest the occurence of a weak conformational change of the active center and/or the formation of non-covalent bonds between the methylphosphonyl residue and side chain groups as a result of the dealkylation reaction.

Formes moleculaires multiples de la butyrylcholinesterase du plasma humain: II. electrophorèse d'affinité des formes C 1, C 3 ET C 4

Multiple molecular forms of human plasma butyrylcholinesterase II.-Study of the C 1, C 3 and C 4 components by means of affinity electrophoresis Affinity electrophoresis has been applied to the analysis of the multiple molecular forms of human plasma cholinesterase allozyme U. A water-soluble p-amino-substituted-phenyltrimethylammoniumpolyacrylamide was synthetized by copolymerization of an unsaturated derivative of the ligand with acrylamide, and entrapped at various concentrations within the matrix of separating gels. Electrophoresis was carried out in these gels, and the relative mobility of the molecular forms of the enzyme was decreased. From the variation of mobility ( R m) as a function of immobilized ligand concentrations, the apparent dissociation constants of monomer (C 1), dimer (C 3) and tetramer (C 4) of phenotype U were calculated. The decrease in mobility was reversed by addition of non-immobilized competitive ligands ( N-methylpyridinium and N-methylacridinium). The appearance of the slopes of R mi -1 vs. concentration does not give sufficient information for determination of the number of anionic binding sites of C 4, but the slight curvature of the plots suggests that bivalent or higher interactions occur when the concentration is sufficiently high. For all three size isomers from a critical ligand concentration, a second zone, named B, appears and intensifies rapidly at the expense of the first zone (A) as the immobilized ligand concentration increases. Among several possible explanations of this phenomenon, it is proposed that the ligand induces a conformational isomerization of the enzymes with a change in affinity ( K D,B < K D,A), and that the interconversion process between the two states B⇆A is slow compared with the ligand-association⇄dissociation steps.

Formes moleculaires multiples de la butyrylcholinesterase du plasma humain I. Parametres moleculaires apparents et ebauche de la structure quaternaire

Apparent molecular parameters (molecular weights, sedimentation constants, partial specific volumes, free electrophoretic mobilities and isoelectric points) of the four molecular forms C-1, C-2, C-3 and C-4 of human plasma butyrylcholinesterase (EC 3.1.1.8) have been demonstrated by polyacrylamide gel electrophoresis methods and centrifugation in sucrose gradient. The C-1 component is the monomeric form of the enzyme ( M r = 84 800 ± 5800). All the forms are partially interconvertible and C-1, C-3, C-4 are size isomers corresponding to the monomer, dimer and tetramer of the enzyme. An estimation of the general shape of these forms attempted from electrophoretic and hydrodynamic parameters suggests that they are prolate ellipsoids. The C-4 component in which the axial ratio is at least equal to 8 appears to be arranged as a dimer of dimers (C-3) 2 in which the two units are associated in a quasi-linear fashion. The C-2 component is composed of C-1 associated with an inactive smaller subunit, which is responsible for its specific electrical properties (mobility and isoelectric point).

Ketamine interaction with neuromuscular blocking agents in the phrenic nerve-hemidiaphragm preparation of the rat.

The influence of ketamine on the directly and indirectly evoked twitch tension and on presynaptic acetylcholine (ACh) release and the interaction of ketamine with neuromuscular blocking agents sere investigated in the isolated phrenic nerve-hemidiaphragm preparation of the rat. Ketarnine, 10 μg/ml, caused an S.65 ± 1.25 SEM (standard error of the mean) percent (p <0.001) increase in indirectly evoked twitch tension hut had no effect on directly stimulated muscle. The same concentration of ketamine decreased presynaptic ACh release from 7.75 ± 0.80 to 5.60 ± 0.54 nmol/g tissue/min (p < 0.005). Ketamine in 4 to 8 μg/ml Concentrations significantly (p <0.05 to 0.001) increased the neuromuscular effects of d-tubocurarine (d-Tc). pancuronium, succinylcholine (SCh), and decamethonium (C10). In the presence of 10 μg/ml ketamine the dose-response regression line of d-Tc and SCh shifted to the left. There was no change in the slope of the d-Tc and SCh regression lines. Ketamine produced 50 percent inhibition of human plasma butyrylcholinesterase at a concentration of 5.2 ± 10−3M and 50 percent inhibition of human red cell acetylcholinesterase at a concentration of 3.3 ± 10−3M. These concentrations are substantially helps plasma levels of ketamine 7.5 minutes after its administration. It is unlikely that ketamine augments the effect of SCh by inhibition of the enzymatic biodegradation of SCh. Our findings suggest that decreased presynaptic ACh release may contribute to the increased effect of neuromuscular blocking agents produced by ketamine in this preparation.

3161. Metabolites of 2,4-toluenediamine: Waring, R. H. & Pheasant, A. E. (1976). Some phenolic metabolites of 2,4-diaminotoluene in the rabbit, rat and guinea-pig. Xenobiotica6, 257

The inhibition of cholinesterases (ChEs) by carbamates includes a carbamylation (inhibition) step, in which the drug transfers its carbamate moiety to the active site of the enzyme and a decarbamylation (activity recovery) step, in which the carbamyl group is hydrolyzed from the enzyme. The carbamylation and decarbamylation kinetics decide the extent and the duration of the inhibition, thus the full characterization of candidate carbamate inhibitors requires the measurement of the kinetic constants describing both steps. Carbamylation and decarbamylation rate constants are traditionally measured by two separate set of experiments, thus making the full characterization of candidate inhibitors time-consuming.In this communication we show that by the analysis of the area under the inhibition-time curve of cholinesterases inhibited by carbamates it is possible to calculate the decarbamylation rate constant from the same data traditionally used to characterize only the carbamylation kinetics, therefore it is possible to obtain a full characterization of the inhibition with a single set of experiments. The characterization of the inhibition kinetics of human and dog plasma butyrylcholinesterase and of human acetylcholinesterase by bambuterol and bambuterol monocarbamate enantiomers was used to demonstrate the validity of the approach. The results showed that the proposed method provides reliable estimations of carbamylation and decarbamylation rate constants thus representing a simple and useful approach to reduce the time required for the characterization of carbamate inhibitors.

The use of echothiophate in myasthenia gravis.

Echothiophate is a potent inhibitor of human plasma butyrylcholinesterase and red cell acetylcholinesterase. In vitro, plasma butyrylcholinesterase was maximally inhibited within two hours, whereas maximal inhibition of red cell acetylcholinesterase occurred after eight hours. In vivo, both enzymes were maximally inhibited in about tour hours. Echothiophate was used for the treatment of subjects whose myasthenia was poorly controlled on short‐acting anticholinesterases. In most, the initial results were good. However, in about one half of the patients it subsequently became necessary to discontinue echothiophate because good control could not be maintained with it. In one third of the patients, however, it was possible to maintain good contral with the drug for over six years. In same instances pyridostigmine supplementatian was necessary to relieve bulbar symptoms. Our experience indicates that echothiophate is most useful in the treatment of severe but stable myasthenia gravis, primarily involving the skeletal muscles. After discontinuation of echothiophate therapy, plasma butyrylcholinesterase and red cell acetylcholinesterase returned to control levels in about one hundred days.

Stability, Toxicity, and Reaction Mechanism with Esterases of Certain Carbamate Insecticides

The stability, activity as inhibitors of acetyl- and butyrylcho-linesterase, and toxicity to house flies, Musca domestica L., and mice were compared for 30 N -alkyl and N,N -dialkylcarbamates. For each type of biological activity a distinctive structural specificity unrelated to stability was observed. The carbamates varied greatly in their inhibitory specificity for the acetyl- and butyrylcholinesterase and in the rate of attaining an equilibrium of cholinesterase, carbamate and acetyl choline during assay. Plasma albumin was found to hydrolyze p -Nitrophenyl N -alkylcarbamates rather than plasma cholinesterase or arylesterase. Chymotrypsin and human plasma butyrylcholinesterase did not react with the carbamates in a similar manner to their reaction with the acetates, ethyl carbonates and dialkyl phosphates derived from the same enols. These results indicate that competition rather than carbamoylation is the primary mechanism of cholinesterase inhibition and presumably toxicity for the N - alkyl and N,N -dialkylcarbamates studied.