Inhibitor Report for: Procainamide

With the view of purifying soluble human brain acetylcholinesterase (AChE) into its separate isoforms, various preparative chromatographic procedures were compared. Chromatofocusing of cerebrospinal fluid (CSF) AChE revealed two major activity peaks, whilst that of caudate nucleus AChE showed one major peak. Both CSF and caudate nucleus AChE eluted at isoelectric points (pI) of between 5.5 and 5.2. Chromatofocusing failed to separate AChE into its individual isoforms, based on qualitative isoelectric focusing. Preparative purification by affinity chromatography showed a better AChE yield with the use of procainamide as a ligand, vis--vis acridinium. Maximum recovery for CSF and caudate nucleus AChE was 10 and 43% using acridinium and procainamide, respectively. Qualitative analysis by SDS-PAGE of affinity-purified AChE revealed four major bands between 50 and 62 kDa, corresponding to the catalytic subunits of AChE as verified by an anti-AChE polyclonal antibody. A size-exclusion column also allowed brain AChE purification, with the latter eluting at a putative molecular mass of 310 kDa. Unfortunately, cation-exchange using the state-of-the-art SMART system failed to separate AChE into its isoforms. AChE aggregation is given as one major obstacle precluding good resolution of isoforms.

Stabilization of fetal bovine serum (FBS) acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AChE) and human butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) (BCHE) by ligands and inhibitors was studied as a function of physical and chemical perturbation. Denaturation of AChE occurred as a binary exponential function in the temperature range studied (50-56 degrees C); the slower fraction progressively diminished as the temperature was increased. Inclusion of ligands or inhibitors stabilized AChE as a function of temperature, ligand concentration and time. The rank order in which ligands stabilized AChE was: edrophonium greater than decamethonium greater than pralidoxime chloride much greater than procainamide. BCHE denaturation was retarded by ligands in the order: decamethonium greater than procainamide greater than edrophonium greater than pralidoxime. A plot of the quotient of the fast/slow ratio against the log of the 50% inhibitory concentration (I50) for ligands providing substantial protection yielded a linear relation, suggesting that these compounds stabilized AChE by a common mechanism involving the anionic site of the active center. Urea-induced cholinesterase denaturation was also retarded by these ligands.

The in vitro effect of procainamide on plasma cholinesterase (PCHE) activity in the plasma of ten normal ASA physical status I patients was studied using a kinetic method. The mean plasma cholinesterase activity without procainamide (control) was 0.90 +/- 0.09 units.ml-1. The dibucaine numbers of all the samples were in the normal range of 78 to 86, indicating normal genotypes. The mean plasma cholinesterase activity, in the presence of procainamide in concentrations of 5.0, 10.0, 20.0 and 40.0 micrograms.ml-1, was reduced to 0.73 +/- 0.04, 0.61 +/- 0.03, 0.45 +/- 0.02, and 0.36 +/- 0.01 units.ml-1, respectively. At therapeutic concentrations of 4 to 12 micrograms.ml-1, procainamide inhibited cholinesterase activity 15 to 30 per cent. The authors also showed that the concentration of procainamide required to inhibit 50 per cent of plasma cholinesterase activity was 20 micrograms.ml-1 (I50). The authors conclude that procainamide when tested in vitro had a statistically significant depressant effect on plasma cholinesterase activity at all the concentrations studied.

Three distinct acetylcholinesterases were detected in the annelid oligochaete Dendrobaena veneta. Two enzymes (alpha, beta), copurified from a Triton-X-100-soluble extract of whole animals by affinity (edrophonium-Sepharose) chromatography, were separately eluted from a Sephadex G-200 column. Gel-filtration chromatography, sedimentation analysis and SDS/PAGE showed the alpha and beta forms to be a globular dimer (110 kDa, 7.0 S) and a hydrophilic monomer (58 kDa, 5.0 S) respectively, both weakly linked to the cell membrane. The third form (gamma), also purified to homogeneity by slower filtration through an edrophonium-Sepharose matrix, proved to be an amphiphilic globular dimer (133 kDa, 7.0 S) with a phosphatidylinositol anchor giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. The alpha acetylcholinesterase showed a fairly low substrate specificity: the beta form hydrolyzed propionylthiocholine at the highest rate and was inactive on butyrylthiocholine; the gamma acetylcholinesterase, showing a marked active-site specificity with differently sized substrates, was likely functional in cholinergic synapses. Studies with inhibitors showed incomplete inhibition of all three acetylcholinesterase by 1 mM eserine and different sensitivity for edrophonium or procainamide. The alpha and beta forms, sensitive to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, were unaffected by tetra(monoisopropyl)-pyrophosphortetramide, while both these agents inhibited the gamma enzyme. All three forms showed excess-substrate inhibition by acetylthiocholine. Enzyme activity was histochemically localized in the nerve ring and its minor branches. Monomeric acetylcholinesterase (beta) is likely the only form present in the ganglionic glial framework.

With the view of purifying soluble human brain acetylcholinesterase (AChE) into its separate isoforms, various preparative chromatographic procedures were compared. Chromatofocusing of cerebrospinal fluid (CSF) AChE revealed two major activity peaks, whilst that of caudate nucleus AChE showed one major peak. Both CSF and caudate nucleus AChE eluted at isoelectric points (pI) of between 5.5 and 5.2. Chromatofocusing failed to separate AChE into its individual isoforms, based on qualitative isoelectric focusing. Preparative purification by affinity chromatography showed a better AChE yield with the use of procainamide as a ligand, vis--vis acridinium. Maximum recovery for CSF and caudate nucleus AChE was 10 and 43% using acridinium and procainamide, respectively. Qualitative analysis by SDS-PAGE of affinity-purified AChE revealed four major bands between 50 and 62 kDa, corresponding to the catalytic subunits of AChE as verified by an anti-AChE polyclonal antibody. A size-exclusion column also allowed brain AChE purification, with the latter eluting at a putative molecular mass of 310 kDa. Unfortunately, cation-exchange using the state-of-the-art SMART system failed to separate AChE into its isoforms. AChE aggregation is given as one major obstacle precluding good resolution of isoforms.

Pseudocholinesterase (ChE) (acylcholineacylhydrolase, EC 3.1.1.8) has been partially purified (about 270-fold) from sheep brain. The procedure included ammonium sulfate fractionation (20-80%), DEAE-Trisacryl M chromatography and procainamide-Sepharose 4B affinity chromatography. The molecular weight of purified ChE was found to be 290,000 by gel filtration. Kinetic properties of the enzyme have been studied using the substrate analogues choline, succinylcholine and benzoylcholine. It was shown that the inhibition was partially competitive.

Acetylcholinesterase (EC 3.1.1.7) and butyrylcholinesterase (EC 3.1.1.8) in human amniotic fluid were estimated in the presence of selective inhibitors. Amniotic fluid cholinesterases (mixture of acetylcholinesterase and butyrylcholinesterase) purified by procainamide-Sepharose affinity chromatography exhibited aryl acylamidase activity which was sensitive to serotonin inhibition (a property of aryl acylamidases associated with both acetyl- and butyrylcholinesterases) and tyramine activation (shown exclusively by aryl acylamidase associated with butyrylcholinesterase). Tyramine activation was unaffected in the presence of the selective acetylcholinesterase inhibitor BW284C51 whereas it was abolished in the presence of the selective butyrylcholinesterase inhibitor ethopropazine, suggesting the presence of both types of aryl acylamidases in amniotic fluid, one associated with acetylcholinesterase and the other associated with butyrylcholinesterase. Butyrylcholinesterase and the associated aryl acylamidase activity in the affinity purified enzyme was selectively immunoprecipitated by a polyclonal antibody raised against human serum butyrylcholinesterase. Estimation of the activity ratio of acetylcholinesterase to butyrylcholinesterase in a few samples of amniotic fluid showed that this could vary depending on the butyrylcholinesterase arising from contaminating blood in the samples. Gel electrophoresis under non-denaturing conditions and enzyme staining showed that butyrylcholinesterase band was detectable on the gel in all the samples whereas acetylcholinesterase band was below detectable levels in normal samples but visible in samples from pregnancies of neural tube defect fetuses. It is suggested that the use of selective cholinesterase inhibitors along with gel electrophoresis and immunoprecipitation studies may be useful in the assessment of cholinesterase activities in human amniotic fluid.

A high-salt soluble form of acetylcholinesterase (AChE) was purified from monkey (Macaca radiata) whole diaphragm by a two step affinity chromatographic procedure using m-aminophenyl trimethylammonium-chloride hydrochloride-Sepharose and procainamide-Sepharose columns. The purified enzyme showed three major protein bands at 80 kDa, 78 kDa and 60 kDa on SDS-gel electrophoresis. [3H]Diisopropyl fluorophosphate ([3H]DFP) labeled enzyme also gave three radioactive peaks corresponding to these three bands. The purified enzyme pretreated with dithiothreitol and subjected to limited trypsin digestion gave a peptide fragment of molecular weight approximately 300 Da showing weak acetylthiocholine hydrolyzing activity as identified by Sephadex G-25 gel filtration. Sequence analysis showed that the active peptide fragment was a tripeptide with the sequence Ala-Gly-Ser. When the purified AChE was labeled with [3H]DFP, digested with trypsin and subjected to Sephadex G-25 chromatography, a radioactive peak that would correspond to the tripeptide fragment was seen. The kinetics, inhibition characteristics and binding characteristics to lectins of the active peptide fragment was compared with the parent enzyme. A synthetic peptide of sequence Ala-Gly-Ser was also found to exhibit acetylthiocholine hydrolyzing activity. The kinetics and inhibition characteristics of the synthetic peptide was similar to those of the peptide derived from the purified enzyme, except that the synthetic peptide was more specific towards acetylthiocholine than butyrylthiocholine. The specific activity (units/mg) of the synthetic peptide was about 29480 times less than that of the purified AChE.

Stabilization of fetal bovine serum (FBS) acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AChE) and human butyrylcholinesterase (acylcholine acylhydrolase, EC 3.1.1.8) (BCHE) by ligands and inhibitors was studied as a function of physical and chemical perturbation. Denaturation of AChE occurred as a binary exponential function in the temperature range studied (50-56 degrees C); the slower fraction progressively diminished as the temperature was increased. Inclusion of ligands or inhibitors stabilized AChE as a function of temperature, ligand concentration and time. The rank order in which ligands stabilized AChE was: edrophonium greater than decamethonium greater than pralidoxime chloride much greater than procainamide. BCHE denaturation was retarded by ligands in the order: decamethonium greater than procainamide greater than edrophonium greater than pralidoxime. A plot of the quotient of the fast/slow ratio against the log of the 50% inhibitory concentration (I50) for ligands providing substantial protection yielded a linear relation, suggesting that these compounds stabilized AChE by a common mechanism involving the anionic site of the active center. Urea-induced cholinesterase denaturation was also retarded by these ligands.

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by co-elution of both peptidase and PsChE activities from procainamide-Sepharose and concanavalin-A--Sepharose affinity chromatographic columns; comigration on polyacrylamide gel electrophoresis; co-elution on Sephadex G-200 gel filtration and coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non-denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr approximately equal to 90,000). Peptidase activity using different peptides showed the release of C-terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N-terminal amino acids. Potent anionic or esterase site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide, ethopropazine, imipramine, diisopropylfluorophosphate, tetra-isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esterase site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate, benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.

The in vitro effect of procainamide on plasma cholinesterase (PCHE) activity in the plasma of ten normal ASA physical status I patients was studied using a kinetic method. The mean plasma cholinesterase activity without procainamide (control) was 0.90 +/- 0.09 units.ml-1. The dibucaine numbers of all the samples were in the normal range of 78 to 86, indicating normal genotypes. The mean plasma cholinesterase activity, in the presence of procainamide in concentrations of 5.0, 10.0, 20.0 and 40.0 micrograms.ml-1, was reduced to 0.73 +/- 0.04, 0.61 +/- 0.03, 0.45 +/- 0.02, and 0.36 +/- 0.01 units.ml-1, respectively. At therapeutic concentrations of 4 to 12 micrograms.ml-1, procainamide inhibited cholinesterase activity 15 to 30 per cent. The authors also showed that the concentration of procainamide required to inhibit 50 per cent of plasma cholinesterase activity was 20 micrograms.ml-1 (I50). The authors conclude that procainamide when tested in vitro had a statistically significant depressant effect on plasma cholinesterase activity at all the concentrations studied.

A simple procedure has been developed for the large scale purification of fetal bovine serum acetylcholinesterase (AChE) (EC 3.1.1.7). The procedure involves two steps: batch adsorption of the AChE from 250 L of serum onto a procainamide affinity Sepharose 4B gel; and analytical procainamide affinity chromatography of the step-1 product. Over 100 mg of AChE was purified in 10 days to apparent homogeneity with this procedure.

Affinity electrophoresis of three purified molecular forms of human plasma cholinesterase (monomer C1, dimer C3, tetramer C4) was carried out in polyacrylamide gels at various total acrylamide concentrations ranging from 3.48 to 9% in a discontinuous buffer system. A water-soluble linear copolymer supporting procainamide, a ligand of the anionic site of cholinesterase, was physically entrapped at various concentrations within the gel network. The combined effects of gel concentration and ligand concentration on the affinity pattern of the three molecular forms were studied. It was found that gel concentration influences the apparent binding activity of their anionic site: The apparent strength of interaction varied with the gel concentration: the denser the gel was, the higher the apparent affinity. The ligand-induced isomerization process was also depending on the gel concentration: the ligand concentration from which each zone is splitting into two moving zones decreased as the total gel concentration increased. These results show that the electrophoretic matrix plays an important role in the affinity process in affinity electrophoresis presumably by controlling kinetic effects: kinetics of protein-ligand complex formation and dissociation reactions, and mass transfer kinetics.

Arsenite is a quasi-irreversible inhibitor of human serum butyrylcholinesterase with a dissociation constant of 0.129 mM at pH 7.4, 25 degrees, 0.067 M phosphate, mu = 0.17 M. The inhibition process is second order with a rate constant of 340 M-1 min-1. The first order rate of dissociation, 0.044 min-1, is unaffected by fluoride but is decreased by substrate. The binding of arsenite and fluoride, as determined by the effect of fluoride on the apparent arsenite-enzyme dissociation constant, is highly anticooperative and may be mutually exclusive. The fluoride-enzyme dissociation constant determined from these experiments is 0.90 mM. The binding of a number of other substances, such as dibucaine, is markedly anticooperative with arsenite binding. The binding of some of these substances is positively cooperative with fluoride binding. The effect can be large; procainamide binds 17 times more strongly in the presence of fluoride. Similarly, the mutual binding of benzoylcholine as substrate and fluoride is cooperative, 30-fold, butyrylthiocholine and fluoride, 21-fold, propionylthiocholine and fluoride, 8.3-fold, and acetylthiocholine and fluoride, only 1.8-fold.

Acetylcholinesterase (EC 3.1.1.7) from fetal bovine serum (FBS) was purified to electrophoretic homogeneity. The procedure involved procainamide affinity chromatography with native FBS, followed by chromatography on Sepharose 6B and DEAE-Sephadex. The acetylcholinesterase was purified approximately 44,000-fold, and 13 mg was obtained corresponding to an overall yield of about 45%. The purified acetylcholinesterase was stable at 4 degrees C for at least 8 weeks but was labile to freezing; however, in 50% glycerol the enzyme was stable at -20 degrees C for at least 12 weeks. FBS acetylcholinesterase exhibited typical substrate inhibition, had a Km of 120 microM, and a turnover number of 5300 s-1 with the substrate acetylthiocholine. The enzyme was highly sensitive to the specific acetylcholinesterase inhibitor 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one. FBS acetylcholinesterase was characterized as a G4 form of acetylcholinesterase and was distinguished from bovine erythrocyte acetylcholinesterase on the basis of lectin gel binding, [3H] Triton X-100 binding, amino acid composition, number of catalytic subunits/molecule, and hydrodynamic properties. FBS acetylcholinesterase had a Stokes radius of 76 A as judged by gel filtration, and from this a molecular weight of 340,000 daltons was calculated. The enzyme had a subunit weight of approximately 83,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; paraoxon titration indicated a relative active site mass of 75,000 daltons. The amino acid composition of FBS acetylcholinesterase was similar to the human erythrocyte acetylcholinesterase (Rosenberry, T. L., and Scoggin, D. M. (1984) J. Biol. Chem. 259, 5643-5652). A monoclonal antibody directed against human erythrocyte acetylcholinesterase, AE-2, (Fambrough, D. M., Engel, A. G., and Rosenberry, T. L. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1078-1082) cross-reacted with FBS acetylcholinesterase.

The following states of purified tetrameric form (C4) 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-procanamide 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-procanamide 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 soman-cholinesterase conjugate (aging) does not seem to induce structural changes detectable in the cross-linkability of lysyle residues at the subunit interfaces and on the 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 occurrence 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.

Human serum aryl acylamidase associated with serum cholinesterase was purified to homogeneity. Evidence for the identity of the two enzymes was based on co-elution profiles, co-purification in the different steps including affinity chromatography with constant ratios of specific activity and percentage recoveries, co-migration on gel electrophoresis, parallel inhibition by typical cholinesterase inhibitors and co-precipitation by antibody raised against the purified enzyme. Human liver aryl acylamidase was partially purified. Based on the elution profiles, purification data, inhibitory characteristics and gel electrophoresis it was concluded that aryl acrylamidase of liver was not associated with liver cholinesterase. More conclusive evidence for the non-association of the liver aryl acylamidase and cholinesterase came from their clear-cut separation on procainamide-Sepharose affinity chromatography. Both the serum and liver aryl acylamidase were compared with the purified erythrocyte aryl acylamidase associated with acetylcholinesterase. While the erythrocyte and serum aryl acylamidases showed some similarities in their sensitivities to amines like serotonin or tryptamine and choline derivatives, the liver enzyme was unaffected by any of these compounds. A notable observation was the activation by tyramine of the serum aryl acylamidase but not the erythrocyte and liver aryl acylamidases. The liver aryl acylamidase also differed from the other two in its relative insensitivity to inhibition by eserine, neostygmine and other cholinesterase inhibitors. Immunodiffusion and immunoprecipitation studies showed that the aryl acylamidases from the liver and erythrocytes were immunologically non-identical with the serum enzyme.