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.     

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.     

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.     

N alpha-dansyl-L-arginine 4-phenylpiperidine amide. A potent and selective inhibitor of horse serum cholinesterase

The relationship between chemical modifications of arginine derivatives and inhibitory activity to horse serum cholinesterase (BuChE) was investigated. It provided a new insight into the topography of the active site of BuChE. 1) BuChE has the hydrophobic binding pocket, the depth of which corresponds to the length of ethylpiperidine. 2) In the opposite side to the hydrophobic binding pocket, BuChE has a certain entity which repulses carboxyl group at the 2-position of piperidine of L-arginine piperidine amide. 3) The P site of BuChE can allow 4-propyl and 4-phenyl group attached to piperidine. Comparison of the results with those of thrombin and trypsin clearly revealed similarities and dissimilarities among BuChE, trypsin, and thrombin in the active site topography, and hence, we introduce a new selective inhibitor for BuChE, N alpha-dansyl-L-arginine 4-phenylpiperidine amide. It inhibits BuChE strongly (Ki = 0.016 microM), whereas it inhibits trypsin, thrombin, plasmin, and glandular kallikrein only weakly and shows actually no inhibition on acetylcholinesterase from the human erythrocyte. In addition, the new inhibitor becomes highly fluorescent when bound with BuChE, indicating that the compound is an ideal probe of the interactions of BuChE as well as a titrant of it.     

Multiple effects of isoproterenol on horse plasma cholinesterase

Isoproterenol inhibits the hydrolysis of butyrylthiocholine by horse plasma cholinesterase, while it stimulates the hydrolysis of p-nitrophenyl butyrate. The inhibition pattern obtained for butyrylthiocholine is consistent with a dimeric model for the enzyme showing negative cooperativity. The kinetics of inhibition point to a dissociative effect of isoproterenol, superimposed on its competitive inhibitory action. The hydrolysis of p-nitrophenyl butyrate is not sensitive to changes in the subunit composition of the enzyme.     

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.     

Structure of human serum cholinesterase

Human cholinesterase has recently been sequenced and cloned. It is a glycoprotein of 4 identical subunits, each subunit containing 9 carbohydrate chains and 3.5 disulfide bonds. Protein folding is likely to be very similar in human cholinesterase and Torpedo acetylcholinesterase. The cholinesterases have no significant sequence homology with the serine proteases and seem to belong to a separate serine esterase family.     

How aliphatic alcohols and pH affect reactivity of horse blood serum cholinesterase at its interaction with organophosphorus inhibitors

There was studied action of aliphatic alcohols (ethanol, propanol, isopropanol, n-butanol, isobutanol, sec-butanol, tert-butanol), and pH on various kinds of serum cholinesterase. At inhibition of the cholinesterase hydrolytic activity under effect of alcohols the key role was played not by the total number of carbon atoms in the alcohol molecule, but by the “efficient length” of the carbohydrate chain. The fact that the presence of alcohols did not affect parameters of reversible inhibition of cholinesterase by onium ions tetramethylammonium and choline allows suggesting the absence of action of solvents on specific sorption of acetylcholine in the enzyme active center. With aid of two sets of hydrophobic organophosphorus inhibitors (OPI) (12 compounds), we have managed to estimate both the degree and the character itself of serum cholinesterase.     

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.     

Molecular biology of human serum cholinesterase

More than 90% of the amino acid sequence of purified human serum cholinesterase has been determined in our laboratory. Purified enzyme was digested with several proteolytic enzymes; the resulting polypeptides were then separated, purified, and sequenced. Optimal sequence regions were identified and used as the basis for the synthesis of three 17-mer oligonucleotide probes. In addition, one long peptide of 58 amino acid residues was selected for construction of two unique sequence oligonucleotide probes of 39-mer and 53-mer; the peptide regions corresponding to the latter are six amino acids apart. The probes have been used to screen a human liver cDNA library and a human genomic library. Several positive clones to both types of probes have been identified. These are being characterized, and some of them have been or are now being sequenced. A high degree of homology in the amino acid sequence of the active center of human serum cholinesterase and that of acetylcholinesterase from the Torpedo fish has been noted. It appears that this region of cholinesterases has been conserved during evolution, and there may be an important, still unrecognized role for serum nonspecific cholinesterase in mammalian metabolism.