Study of hydrolysis of aminoalcohol ethers, phenol and choline under the action of horse blood serum cholinesterase]

Hydrolysis of ethers of saturated and unsaturated alcohols and ethers, e.g. phenol and choline, under the action of horse blood serum cholinesterase, was studied. The reactivity towards enzymatic hydrolysis is decreased due to a greater length of the chain in the alcohol residue of the benzoic acid aminoethers; at nCH2 = 4 the compound is a poor substrate. An increase in nydrophobicity of the acyl residue of the ether molecule also leads to a decrease in the Vmax and Km values. In case of cholinesterase substrates, an increase in the molecule hydrophobicity results in an increase of its non-productive absorption on the active surface of the enzyme, which decreases its hydrolysis. Aminobutynol benzoates are hydrolyzed by cholinesterase more rapidly as compared to the ethers of corresponding aminobutanols and their homologs.     

Isoproterenol inhibition of horse serum cholinesterase is connected with subunit dissociation

Tetrameric cholinesterase from horse serum undergoes concentration-dependent dissociation. The dimer is highly stable so that even on SDS polyacrylamide gels subunit dissociation to the 80-kDa polypeptide chains is incomplete. Glutaraldehyde cross-linking confirms this finding, giving rise to a tetramer: dimer ratio of approximately 1:1. The beta-adrenergic agent isoproterenol acts as an inhibitor of the enzyme with respect to butyrylthiocholine hydrolysis; inhibition kinetics point to a dissociative effect of the ligand as the underlying mechanism (S?ylemez, Z. & Ozer, I. (1985) Comp. Biochem. Physiol. 81c, 433-437). Evidence from sedimentation analysis confirms this hypothetical mechanism: the sedimentation coefficient in the presence of saturating concentrations of both the substrate butyrylthiocholine and the inhibitor isoproterenol shows a 35 +/- 5% decrease; in high speed sedimentation equilibria the weight average molecular mass is shifted from the tetramer (Mr = 312 +/- 12 kDa) to the dimer (Mr = 160 +/- 10 kDa). The transition is complete at isoproterenol concentrations below saturation. Applying glutaraldehyde cross-linking to monitor the particle distribution at varying isoproterenol concentrations confirms the change in quaternary structure in a qualitative way. Enzyme concentrations applied in the present experiments are in the range of the concentration of cholinesterase in horse serum. Therefore the dissociative mechanism of isoproterenol on the enzyme may be of biological significance.     

On the inhibition of cholinesterase by D-tubocurarine

Some investigation in this laboratory pointed to an unexpectedly slow inhibition of cholinesterase by D-tubocurarine, occurring in addition to a typically instantaneous inhibition. In order to elucidate this phenomenon, the hydrolysis of butyrylthiocholine catalyzed by cholinesterase was recorded, in the absence and presence of D-tubocurarine, on a stopped-flow apparatus. Experimental results were analyzed by classical kinetic methods and by means of mathematical modeling. It was found that the inhibition is of a double character, consisting of an instantaneous phase and a slow one occurring in a minute time scale. It seems that the action of D-tubocurarine is a consequence of an instantaneous binding of D-tubocurarine to a peripheral site, followed by a relatively slow conformational transition in the enzyme.     

Active site kinetics of horse serum cholinesterase

Horse serum Cholinesterase hydrolyzes choline and p-nitrophenol esters at different rates. Deacylation and acylation seem to be the rate-limiting step for charged and uncharged substrates respectively. Activation energy is similar for the acetic, propionic, and butyric esters of thiocholine, but it is higher for p-nitrophenylpropionate. Inhibition by the tetramethyl-ammonium ion is competitive. Tetraethyl-, tetrapropyl-, and tetrabutyl-ammonium ions are mixed-type inhibitors. The pH studies demonstrated the existence of a residue, pK = 6.33, involved in catalysis.     

The purification of cholinesterase from horse serum

A relatively simple method is described by which cholinesterase was purified about 19000-fold starting from horse serum. Typically 20 litres of serum were processed to yield 15-18mg of electrophoretically pure cholinesterase in the form of an active salt-free dry powder. The method included two stages: fractionation with (NH(4))(2)SO(4) and ion-exchange chromatography. The (NH(4))(2)SO(4) stage included, in principle, the acid (pH3) step of the Strelitz (1944) procedure. The step took advantage of the stabilizing effect that 33%-satd. (NH(4))(2)SO(4) has on cholinesterase activity at pH3 and it is recognized that in the absence of (NH(4))(2)SO(4) the enzyme is rapidly destroyed at pH3. Cholinesterase was significantly more stable to pH3.0 at 2 degrees C than at 24 degrees C, and the acid step was done at both temperatures. The specific activities of the final products obtained by way of acid steps were the same at either temperature, thus indicating that the step has not harmed the enzyme active sites. The product from the first two stages was purified over 18000-fold and was 85-90% cholinesterase. The remaining impurities were removed by preparative gel electrophoresis. The product was about 40% more active and contained 40% more active sites per unit weight than electrophoretically pure cholinesterase prepared from partially purified commercial starting material. Although the number of active sites per molecule was not determined with certainty, a value of at least 3 and possibly 4 was indicated. The partial specific volumes were determined with a precision density meter, on the ultracentrifuge and from the amino acid and carbohydrate composition. The values by these independent methods were 0.688, 0.71 and 0.712ml/g, respectively. The amino acid and carbohydrate composition was determined. The cholinesterase contained 17.4% carbohydrate including 3.2% N-acetylneuraminic acid.     

Dibucaine inhibition of serum cholinesterase

The dibucaine number (DN) was determined for serum cholinesterase (EC 3.1.1.8, SChE) in plasma samples. The ones with a DN of 79-82 were used, because they had the "usual" SChE variant. The enzyme was assayed colorimetrically by the reaction of 5,5'-dithiobis-[2-nitrobenzoic acid] (DTNB) with the free sulfhydryl groups of thiocholine that were produced by the enzyme reaction with butrylthiocholine (BuTch) or acetylthiocholine (AcTch) substrates, and measured at 412 nm. Dibucaine, a quaternary ammonium compound, inhibited SChE to a minimum within 2 min in a reversible manner. The inhibition was very potent. It had an IC(50) of 5.3 microM with BuTch or 3.8 microM with AcTch. The inhibition was competitive with respect to BuTch with a K(i) of 1.3 microM and a linear-mixed type (competitive/noncompetitive) with respect to AcTch with inhibition constants, K(i) and K(I) of 0.66 and 2.5 microM, respectively. Dibucaine possesses a butoxy side chain that is similar to the butryl group of BuTch and longer by an ethylene group from AcTch. This may account for the difference in inhibition behavior. It may also suggest the existence of an additional binding site, other than the anionic binding site, and of a hydrophobic nature.     

Genetic control of cholinesterase activity in horse serum

The cholinesterase activity in horse serum was shown to be controlled by four co-dominant autosomal alleles, Ch ^A, Ch ^B , Ch ^c and Ch ^D. The respective alleles controlled an activity of approximately 40, 76, 126 and 155 units. Nine phenotypes with different enzyme levels were distinguished. Family data with 11 stallions, 108 mares and 128 offspring were consistent with the genetic theory. The gene frequencies in the North Swedish horse breed were 0.32, 0.42, 0.13 and 0.13, respectively.     

Non-quantal acetylcholine release after cholinesterase inhibition in vivo.

After anticholinesterase treatment in vivo, depolarization of the postsynaptic muscle fibre membrane by about 4 mV develops due to non-quantally released acetylcholine from the motor nerve terminal. This conclusion was supported by experiments with the curarization of diaphragm slices from anticholinesterase treated mice during intracellular microelectrode recordings.     

Determination of serum cholinesterase activity and dibucaine numbers by an amperometric choline sensor

Serum cholinesterase activity and the dibucaine numbers have been determined by using a hydrogen peroxide electrode and the enzyme choline oxidase immobilized on a nylon net. The analysis procedure is extremely simple and very fast allowing 30 cholinesterase determinations per hour. Both cholinesterase activity and dibucaine number measurements could be performed in 5 min and by using serum samples of only 10 microliters. When used in sera the probe showed no interference from electroactive compounds present in blood, and also showed good stability and reproducibility. These features make this sensor appropriate for continuous extracorporeal circuit blood monitoring of succinylcholine during surgery.     

The hydrolysis of rac-glycerol 1-monodecanoate by serum butyryl cholinesterase

Butyryl cholinesterase from horse and human sera catalyzed the hydrolysis of monoacylglycerols containing fatty acids varying in chain length from 8 to 12 carbons; maximum activity was obtained with rac-glycerol 1-monodecanoate as substrate. Neither the triacylglycerols of these fatty acids nor the monoacylglycerols of longer chain length fatty acids were hydrolyzed at measurable rates in the system used. The enzyme was eserine sensitive and indistinguishable from butyryl cholinesterase as judged by purification, response to the several inhibitors tested, and heat inactivation. Data from mixed substrate experiments suggest a possible effector role for butyryl choline in accelerating the rate of rac-glycerol 1-monodecanoate hydrolysis. Fatty acid released during the course of rac-glycerol 1-monodecanoate hydrolysis may irreversibly inactivate the enzyme.