Human Serum Pseudocholinesterase Research Articles

Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues.

To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1.1.8) and Torpedo electric organ "true" acetylcholinesterase (AcChoEase; EC 3.1.1.7). Using these probes, we isolated several cDNA clones from lambda gt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A)+ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These findings demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.

A peptidase activity exhibited by human serum pseudocholinesterase.

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.

Use of synthetic oligodeoxynucleotide probes for the isolation of a human cholinesterase cDNA clone.

Cholinesterases are serine esterases that rapidly hydrolyze the neurotransmitter acetylcholine. In humans, cholinesterases exhibit extensive polymorphism in terms of their substrate specificity, sensitivity to selective inhibitors, hydrophobicity, and cellular as well as subcellular localization. It is not yet known whether the various cholinesterase forms originate from different genes or are products of posttranscriptional and posttranslational processing. The extent to which these enzyme forms are homologous in their amino acid sequence is also not known. However, a consensus organophosphate-binding hexapeptide sequence Phe-Gly-Glu-Ser-Ala-Gly was found both in "true" acetylcholinesterase from the electric organ of Torpedo [McPhee-Quigley et al: J Biol Chem 260:12185-12189, 1985] and in "pseudocholinesterase" (butyrylcholinesterase) from human serum [Lockridge: "Cholinesterases--Fundamental and Applied Aspects." New York: de Gruyter pp 5-12, 1984], suggesting that this region in the protein is conserved in all cholinesterases. Based on this common sequence, we prepared synthetic oligodeoxynucleotides and used them as labeled probes to screen a cDNA library from fetal human brain mRNA, cloned in lambda gt10 phages. A cDNA clone of 770 nucleotides in length was isolated. It contains an open reading frame terminating with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala, which includes the consensus hexapeptide used for designing the DNA probe. Furthermore, the sequence of this 12-amino acid peptide is identical to the sequence reported for the organophosphate binding site of human serum pseudocholinesterase [Lockridge: "Cholinesterases--Fundamental and Applied Aspects." New York: de Gruyter, pp 5-12, 1984]. These findings confirm that the isolated clone is indeed part of a human cholinesterase cDNA.

Chemical modification of the bifunctional human serum pseudocholinesterase. Effect on the pseudocholinesterase and aryl acylamidase activities.

The effect of chemical modification on the pseudocholinesterase and aryl acylamidase activities of purified human serum pseudocholinesterase was examined in the absence and presence of butyrylcholine iodide, the substrate of pseudocholinesterase. Modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, diethylpyrocarbonate and trinitrobenzenesulfonic acid caused a parallel inactivation of both pseudocholinesterase and aryl acylamidase activities that could be prevented by butyrylcholine iodide. With phenylglyoxal and 2,4-pentanedione as modifiers there was a selective activation of pseudocholinesterase alone with no effect on aryl acylamidase. This activation could be prevented by butyrylcholine iodide. N-Ethylmaleimide and p-hydroxy-mercuribenzoate when used for modification did not have any effect on the enzyme activities. The results suggested essential tryptophan, lysine and histidine residues at a common catalytic site for pseudocholinesterase and aryl acylamidase and an arginine residue (or residues) exclusively for pseudocholinesterase. The use of N-acetylimidazole, tetranitromethane and acetic anhydride as modifiers indicated a biphasic change in both pseudocholinesterase and aryl acylamidase activities. At low concentrations of the modifiers a stimulation in activities and at high concentrations an inactivation was observed. Butyrylcholine iodide or propionylcholine chloride selectively protected the inactivation phase without affecting the activation phase. Protection by the substrates at the inactivation phase resulted in not only a reversal of the enzyme inactivation but also an activation. Spectral studies and hydroxylamine treatment showed that tyrosine residues were modified during the activation phase. The results suggested that the modified tyrosine residues responsible for the activation were not involved in the active site of pseudocholinesterase or aryl acylamidase and that they were more amenable for modification in comparison to the residues responsible for inactivation. Two reversible inhibitors of pseudocholinesterase, namely ethopropazine and imipramine, were used as protectors during modification. Unlike the substrate butyrylcholine iodide, these inhibitors could not protect against the inactivation resulting from modification by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide and trinitrobenzenesulfonic acid. But they could protect against the activation of pseudocholinesterase and aryl acylamidase by low concentrations of N-acetylimidazole and acetic anhydride thereby suggesting that the binding site of these inhibitors involves the non-active-site tyrosine residues.

Effect of para substituents of 2-(N,N-dimethylamino)ethyl phenyl phenylphosphonates on inhibition of human pseudocholinesterase.

Human serum pseudocholinesterase inhibition by nineteen of the title phosphonates was studied and the effect of para substituents of the phenoxy and phenyl groups on inhibition rates was compared. The effect of substituents of the phenoxy group on the reaction rate is in some cases even 60 times as great as the effect of substituents of the phenyl group. Logarithms of rate constants correlate linearly with sigma po substituent constants of the phenoxy group, but no linear dependence is observed for para substituents of the phenyl group. The role of the apicophilicity of substituents of phosphorus in the enzyme--inhibitor complex is discussed.

The relationships between human serum pseudocholinesterase, lipoproteins, and apolipoproteins (APOHDL)

During recent years there has been an increasing number of studies concerning metabolizing enzymes during lipoprotein catabolism. It is well documented that lecithin-cholesterol acyltransferase (LCAT, ED 2.3.1.43) and lipoprotein lipase (EC 3.1.1.34) take part with these reactions (1). In addition to these enzymes there has been evidence that pseudocholinesterase (EC 3.1.1.8.) is also associated with lipid metabolism (2). A relationship between serum pseudocholinesterase and low density lipoprotein (LDL) has been shown on the basis of ultrasonication studies (3) and after artifical induction of hyperlipidemia in vitro and in vivo (4). Because pseudocholinesterase activity has been previously measured only in whole serum and in DLD, our purpose has been to find out if there is any activity in other lipoprotein fractions. In addition to whole enzyme activity we investigated the distribution of pseudocholinesterase (PCE) isoenzymes in different fractions. As previously reported, LCAT and lipoprotein lipase need as cofactor, a specific apolipoprotein (5). We also studied a possible connection between HDL (high density lipoprotein) apolipoproteins (apoA-I, A-II, E, and total apoC) and PCE activity.

Atypical and inhibited human serum pseudocholinesterase. A titrimetric method for differentiation.

A method is described for differentiating between lowered pseudocholinesterase levels due to exposure to organophosphate insecticides and those due to a genetically determined deficiency. The method employs a pH-Stat, acetylcholine iodide as the substrate, and dibucaine hydrochloride as an inhibitor. A histogram of typical dibucaine numbers (percent inhibition) and a discussion of the relevance of the concept of dibucaine number to exposure is presented.

Evidence for different "silent genes" in the human serum pseudocholinesterase polymorphism.

Annals of the New York Academy of SciencesVolume 151, Issue 1 p. 540-544 EVIDENCE FOR DIFFERENT “SILENT GENES” IN THE HUMAN SERUM PSEUDOCHOLINESTERASE POLYMORPHISM H. W. Goedde, H. W. Goedde Department of Biochemical Genetics, Institute of Human Genetics and Anthropology, University of Freiburg/Br., GermanySearch for more papers by this authorK. Altland, K. Altland Department of Biochemical Genetics, Institute of Human Genetics and Anthropology, University of Freiburg/Br., GermanySearch for more papers by this author H. W. Goedde, H. W. Goedde Department of Biochemical Genetics, Institute of Human Genetics and Anthropology, University of Freiburg/Br., GermanySearch for more papers by this authorK. Altland, K. Altland Department of Biochemical Genetics, Institute of Human Genetics and Anthropology, University of Freiburg/Br., GermanySearch for more papers by this author First published: June 1968 https://doi.org/10.1111/j.1749-6632.1968.tb11913.xCitations: 22AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume151, Issue1June 1968Pages 540-544 RelatedInformation

Inhibition of cholinesterases by fluoride in vitro.

1. Series of colorimetric dynamic assays allowed the study of the inhibition of cholinesterases by F(-) ions in vitro, by using, as sources of enzyme, whole human blood, human serum, homogenized rat brain and two preparations of red blood cells (human and bovine) whose enzymic purity was ascertained. 2. The first evidence of inhibition of human serum pseudocholinesterase by fluoride was noticed at 15-25mum-fluoride. Ten times as much fluoride was needed to start inhibition of acetylcholinesterase of the red blood cells. 3. The action of fluoride on the enzymic reaction was immediate. The reversibility of the inhibition was shown by dialysis and dilution. 4. Kinetic measurements showed that the inhibition under study was not dependent on the substrate concentration and was of the uncompetitive type, similar to that observed in the presence of a heavy metal (cadmium). 5. The activity of serum cholinesterase did not change in the absence of Mg(2+) and Ca(2+) ions. Fluoride was shown to inhibit the enzyme in the absence of these ions as well as of phosphate. 6. Fluoride could inhibit cholinesterases in the presence of three different substrates and had no action on the non-enzymic hydrolysis. 7. It is thought that the halide is bound reversibly to the enzyme molecule, with the probable exclusion of the active site, but no firm conclusion could be reached on this point.

Progress in Medical Genetics

This represents the third volume of an excellent series covering a wide variety of timely subjects in medical genetics. Actually, the nature of the material is such that since the publication of this volume new developments have already occurred in some areas. It is to the credit of the editors and contributing authors that each topic is presented with a background sufficiently broad to provide an excellent foundation for understanding future developments in the field. The nature of the gene, the genetic code, and the mechanism of protein synthesis are presented in chapter 1 by Strauss in the extremely comprehensible and yet sophisticated style which characterized his earlier text on this subject. In chapter 2 Motulsky emphasizes to clinicians and geneticists the importance of pharmacogenetics. Lehmann and Lidell describe an excellent experimental model for the study of pharmacogenetics in their treatment of human serum pseudocholinesterase. Amos presents transplantation genetics and

Frequency of atypical pseudocholinesterase in British and Mediterranean populations.

CONSIDEBABLE interest has been taken over the past twenty years in the level of human serum pseudo-cholinesterase, because it was known that it was altered ina number of pathological conditions. It has been recognized, however, in the past ten years that considerable differences may also exist in healthy people, and that this difference is genetically determined1. Several variants of the enzyme are now known2–5, but the variants which give rise to the most frequently found phenotypes are the ‘typical’ and ‘atypical’ enzyme of Kalow and Genest2. These can be distinguished by their sensitivity to the pseudocholinesterase inhibitor dibucaine. Under given conditions the ‘typical’ enzyme is inhibited by about 80 per cent (‘Dibucaine Number’ or DN = 80), and the ‘atypical’ enzyme by about 20 per cent (DN = 20). The three phenotypes, ‘typical’ homozygotes, ‘atypical’ homozygotes and heterozygotes show a DN of about 80, 20 and 60 respectively. Kalow and Gunn6 found the gene frequency of the ‘atypical’ gene to be 0.0188 in a healthy Canadian population. ‘Atypical’ homozygotes would therefore be expected at a frequency of 1 in 3,000. This is of practical importance because they respond to the usually short acting, and in modern anaesthetics widely used muscle relaxant suxamethonium with a prolonged and dangerous apnœa, the normally short action of the drug being due to its rapid destruction by the ’typical‘ serum pseudocholinesterase. We had the impression that this anaesthetic hazard was more often encountered in persons of Mediterranean origin than in British patients. We have, therefore, determined the gene frequency of the ‘atypical’ gene in British and various Mediterranean populations by measuring the DN. The results are shown in Table 1. It may be noted that only heterozygotes and no ‘atypical’ homozygotes were discovered.