Cynthia F. Bartels

Tissue distribution of human acetylcholinesterase and butyrylcholinesterase messenger RNA

Cholinesterase inhibitors occur naturally in the calabar bean (eserine), green potatoes (solanine), insect-resistant crab apples, the coca plant (cocaine) and snake venom (fasciculin). There are also synthetic cholinesterase inhibitors, for example man-made insecticides. These inhibitors inactivate acetylcholinesterase and butyrylcholinesterase as well as other targets. From a study of the tissue distribution of acetylcholinesterase and butyrylcholinesterase mRNA by Northern blot analysis, we have found the highest levels of butyrylcholinesterase mRNA in the liver and lungs, tissues known as the principal detoxication sites of the human body. These results indicate that butyrylcholinesterase may be a first line of defense against poisons that are eaten or inhaled.

The Butyrylcholinesterase K-Variant Shows Similar Cellular Protein Turnover and Quaternary Interaction to the Wild-Type Enzyme

Abstract: A recent study has linked the butyrylcholinesterase (BChE) K‐variant and the apolipoprotein ε4 isoform to late‐onset Alzheimer’s disease. These findings have been controversial and have led us to examine the differences between wild‐type and K‐variant BChE in enzyme activity, protein stability, and quaternary structure. J‐variant BChE (E497V/A539T) was also studied because it is associated with the K‐variant mutation. The K‐variant mutation (A539T) is located in the C‐terminal tetramerization domain. Wild‐type, K‐variant, and J‐variant BChE were expressed in Chinese hamster ovary cells and purified. The purified enzymes had similar binding affinity (Km) values and catalytic rates for butyrylthiocholine and benzolycholine. In pulse‐chase studies the K‐variant, J‐variant, and wild‐type BChE were degraded rapidly within the cell, with a half‐time of ∼ 1.5 h. Less than 5% of the intracellular BChE was exported. The C‐terminal peptide containing the K‐variant mutation interacted with itself as strongly as did the wild‐type peptide in the yeast two‐hybrid system. Both K‐variant and wild‐type BChE assembled into tetramers in the presence of poly‐L‐proline or the proline‐rich attachment domain of the collagen tail. The native K‐variant BChE in serum showed the same proportion of tetramers as the native serum wild‐type BChE. We conclude that the K‐variant BChE is similar to wild‐type BChE in enzyme activity, protein turnover, and tetramer formation.

Substrate activation in acetylcholinesterase induced by low pH or mutation in the π-cation subsite

Substrate inhibition is considered a defining property of acetylcholinesterase (AChE), whereas substrate activation is characteristic of butyrylcholinesterase (BuChE). To understand the mechanism of substrate inhibition, the pH dependence of acetylthiocholine hydrolysis by AChE was studied between pH 5 and 8. Wild-type human AChE and its mutants Y337G and Y337W, as well as wild-type Bungarus fasciatus AChE and its mutants Y333G, Y333A and Y333W were studied. The pH profile results were unexpected. Instead of substrate inhibition, wild-type AChE and all mutants showed substrate activation at low pH. At high pH, there was substrate inhibition for wild-type AChE and for the mutant with tryptophan in the pi-cation subsite, but substrate activation for mutants containing small residues, glycine or alanine. This is particularly apparent in the B. fasciatus AChE. Thus a single amino acid substitution in the pi-cation site, from the aromatic tyrosine of B. fasciatus AChE to the alanine of BuChE, caused AChE to behave like BuChE. Excess substrate binds to the peripheral anionic site (PAS) of AChE. The finding that AChE is activated by excess substrate supports the idea that binding of a second substrate molecule to the PAS induces a conformational change that reorganizes the active site.

A Naturally Occurring Genetic Variant in the Human Chorionic Gonadotropin-β Gene 5 Is Assembly Inefficient1

The hCGβ gene family is composed of six homologous genes linked in tandem repeat on chromosome 19; the order of the genes is 7, 8, 5, 1, 2, and 3. Previous studies have shown that hCGβ gene 5 is highly expressed during the first trimester of pregnancy. The purpose of our study was to identify naturally occurring polymorphisms in hCGβ gene 5 and determine whether these alterations affected hCG function. The data presented here show that hCGβ gene 5 was highly conserved in the 334 asymptomatic individuals and 41 infertile patients examined for polymorphisms using PCR followed by single stranded conformational polymorphism analysis. Most of the polymorphisms detected were either silent or located in intron regions. However, one genetic variant identified in β gene 5 exon 3 was a G to A transition that changed the naturally occurring valine residue to methionine in codon 79 (V79M) in 4.2% of the random population studied. The V79M polymorphism was always linked to a silent C to T transition in codon 82 (tyros...

Determination of the DNA sequences of acetylcholinesterase and butyrylcholinesterase from cat and demonstration of the existence of both in cat plasma

Cat serum contains 0.5 mg/L of butyrylcholinesterase (BChE, EC 3.1.1. 8) and 0.3 mg/L of acetylcholinesterase (AChE, EC 3.1.1.7); this can be compared with 5 mg/mL and < 0.01 mg/L, respectively, in human serum. Cat BChE differed from human BChE in the steady-state turnover of butyrylthiocholine, having a 3-fold higher k(cat) and 2-fold higher K(m) and K(ss) values. Sequencing of the cat BCHE cDNA revealed 70 amino acid differences between cat and human BChE, three of which could account for these kinetic differences. These amino acids, which were located in the region of the active site, were Phe398Ile, Pro285Leu, and Ala277Leu (where the first amino acid was found in human and the second in cat). Sequencing genomic DNA for cat and human ACHE demonstrated that there were 33 amino acid differences between the cat and human AChE enzymes, but that there were no differences in the active site region. In addition, a polymorphism in intron 3 of the human ACHE gene was detected, as well as a silent polymorphism at Y116 of the cat ACHE gene.

Endogenous butyrylcholinesterase in SV40 transformed cell lines: COS-1, COS-7, MRC-5 SV40, and WI-38 VA13

SummaryComparison of proteins expressed by SV40 transformed cell lines and untransformed cell lines is of interest because SV40 transformed cells are immortal, whereas untransformed cells senesce after about 50 doublings. In MRC-5 SV40 cells, only seven proteins have previously been reported to shift from undetectable to detectable after transformation by SV40 virus. We report that butyrylcholinesterase is an 8th protein in this category. Butyrylcholinesterase activity in transformed MRC-5 SV40 cells increased at least 150-fold over its undetectable level in MRC-5 parental cells. Other SV40 transformed cell lines, including COS-1, COS-7, and WI-38 VA13, also expressed endogenous butyrylcholinesterase, whereas the parental, untransformed cell lines, CV-1 and WI-38, had no detectable butyrylcholinesterase activity or mRNA. Infection of CV-1 cells by SV40 virus did not result in expression of butyrylcholinesterase, showing that the butyrylcholinesterase promoter was not activated by the large T antigen of SV40. We conclude that butyrylcholinesterase expression resulted from events related to cell immortalization and did not result from activation by the large T antigen.

Acetylcholinesterase and Butyrylcholinesterase of Cat

Domestic cat (Felis domesticus) pituitary cDNA and leukocyte genomic DNA were amplified by PCR and sequenced for the protein coding regions of butyrylcholinesterase (BChE) and acetylcholinesterase (AChE). ~2 kb of BCHE cDNA and ~4 kb of the ACHE gene with introns were sequenced. Bengal tiger (Panthera tigris) pituitary cDNA was sequenced for BCHE. Cat and human comparison showed amino acid identities of 94% (AChE) and 88% (BChE). Inhibition assays with specific AChE or BChE inhibitors (BW284C51, edrophonium, ethopropazine, bambuterol, iso-OMPA, dibucaine and Ro 2-0683) showed that cat plasma Cholinesterase activity was 60% BChE and 40% AChE and tiger plasma Cholinesterase activity was 77% BChE and 23% AChE. Gel electrophoresis with selective staining and inhibitor treatment identified AChE, BChE, paraoxonase, and carboxyleserase in cat and tiger plasma. A monomer of AChE was identified in human serum. An alu repeat containing a guanosine/adenine polymorphism [allelic frequency 0.62 (guanidine) and 0.38 (adenosine)] was found in human ACHE intron 3.

A Comparison of Blood Cholinesterase Activities, Pyridostigmine Inhibition of Red Cell Acetylcholinesterase, and Butyrylcholinesterase Phenotypes in Gulf War Veterans and Normal Controls

A study was undertaken to determine if abnormally low blood cholinesterase (ChE) activity, abnormal red cell acetylcholinesterase (RBC-AChE) pyridostigmine (PB) inhibition kinetics, and/or unusually high frequencies of the atypical phenotype of plasma butyrylcholinesterase (HS-BChE) could explain some of the symptoms exhibited by Gulf War veterans or represent a risk factor for adverse effects after pyridostigmine exposure.

Peripheral Anionic Site of Wild-Type and Mutant Human Butyrylcholinesterase

Although the existence of a peripheral “anionic” site (PAS) on the surface of AChE has long been demonstrated, there was no direct evidence for a PAS on BuChE. However, structural similarities between the two enzymes and some kinetic complexities of BuChE suggested that the mouth of the BuChE active site gorge may possess PAS components.