Hinrichs SH

References (17)

Title : Human butyrylcholinesterase in Cohn fraction IV-4 purified in a single chromatography step on Hupresin - Schopfer_2023_PLoS.One_18_e0280380
Author(s) : Schopfer LM , David E , Hinrichs SH , Lockridge O
Ref : PLoS ONE , 18 :e0280380 , 2023
Abstract : Protection from the toxicity of nerve agents is achieved by pretreatment with human butyrylcholinesterase (BChE). Current methods for purifying large quantities of BChE from frozen Cohn fraction IV-4 produce 99% pure enzyme, but the yield is low (21%). Our goal was to simplify the purification procedure and increase the yield. Butyrylcholinesterase was extracted from frozen Cohn fraction IV-4 in 10 volumes of water pH 6. The filtered extract was pumped onto a Hupresin affinity column. The previously utilized anion exchange chromatography step was omitted. Solvent and detergent reagents used to inactivate lipid enveloped virus, bacteria and protozoa did not bind to Hupresin. BChE was eluted with 0.1 M tetramethylammonium bromide in 20 mM sodium phosphate pH 8.0. BChE protein was concentrated on a Pellicon tangential flow filtration system and demonstrated to be highly purified by mass spectrometry. A high pump rate produced protein aggregates, but a low pump rate caused minimal turbidity. Possible contamination by prekallikrein and prekallikrein activator was examined by LC-MS/MS and by a chromogenic substrate assay for kallikrein activity. Prekallikrein and kallikrein were not detected by mass spectrometry in the 99% pure BChE. The chromogenic assay indicated kallikrein activity was less than 9 mU/mL. This new, 1-step chromatography protocol on Hupresin increased the yield of butyrylcholinesterase by 200%. The new method significantly reduces production costs by optimizing yield of 99% pure butyrylcholinesterase.
ESTHER : Schopfer_2023_PLoS.One_18_e0280380
PubMedSearch : Schopfer_2023_PLoS.One_18_e0280380
PubMedID: 36638134

Title : Purification of human butyrylcholinesterase from frozen Cohn fraction IV-4 by ion exchange and Hupresin affinity chromatography - Schopfer_2019_PLoS.One_14_e0209795
Author(s) : Schopfer LM , Lockridge O , David E , Hinrichs SH
Ref : PLoS ONE , 14 :e0209795 , 2019
Abstract : Human butyrylcholinesterase (HuBChE) is being developed as a therapeutic for protection from the toxicity of nerve agents. An enriched source of HuBChE is Cohn fraction IV-4 from pooled human plasma. For the past 40 years, purification of HuBChE has included affinity chromatography on procainamide-Sepharose. The present report supports a new affinity sorbent, Hupresin, for purification of HuBChE from Cohn fraction IV-4. Nine batches of 70-80 kg frozen Cohn fraction were extracted with water, filtered, and chromatographed on 30 L of Q-Ceramic ion exchange sorbent at pH 4.5. The 4% pure Q-eluent was pumped onto 4.2 L Hupresin, where contaminants were washed off with 0.3 M NaCl in 20 mM sodium phosphate pH 8.0, before 99% pure HuBChE was eluted with 0.1 M tetramethylammonium bromide. The average yield was 1.5 g of HuBChE from 80 kg Cohn paste. Recovery of HuBChE was reduced by 90% when the paste was stored at -20 degrees C for 1 year, and reduced 100% when stored at 4 degrees C for 24h. No reduction in HuBChE recovery occurred when paste was stored at -80 degrees C for 3 months or 3 years. Hupresin and procainamide-Sepharose were equally effective at purifying HuBChE from Cohn fraction. HuBChE in Cohn fraction required 1000-fold purification to attain 99% purity, but 15,000-fold purification when the starting material was plasma. HuBChE (P06276) purified from Cohn fraction was a 340 kDa tetramer of 4 identical N-glycated subunits, stable for years in solution or as a lyophilized product.
ESTHER : Schopfer_2019_PLoS.One_14_e0209795
PubMedSearch : Schopfer_2019_PLoS.One_14_e0209795
PubMedID: 30625168

Title : Heat pretreatment eliminates spurious butyrylcholinesterase enhancement of endotoxin levels in the kinetic chromogenic assay - Brawner_2016_Chem.Biol.Interact_249_19
Author(s) : Brawner A , Hinrichs SH , Larson MA , Lockridge O
Ref : Chemico-Biological Interactions , 249 :19 , 2016
Abstract : The kinetic chromogenic endotoxin assay measures the release of p-nitroaniline from the chromogenic peptide substrate Ac-IEAR-pNA. As part of our project to purify large quantities of human butyrylcholinesterase (HuBChE), we evaluated pure HuBChE for endotoxin levels. We found that HuBChE contributed up to 90% of the yellow p-nitroaniline product in a standard endotoxin assay through the catalytic hydrolysis of Ac-IEAR-pNA with a rate constant of 0.016 min-1 and a Km of 2.9 mM in potassium phosphate buffer pH 7.0 at 24 degrees C. Thus, endotoxin concentrations for native BChE are artificially high in the kinetic chromogenic assay. Destruction of HuBChE catalytic activity by boiling yields endotoxin concentrations that more accurately reflect the endotoxin concentration in purified HuBChE preparations.
ESTHER : Brawner_2016_Chem.Biol.Interact_249_19
PubMedSearch : Brawner_2016_Chem.Biol.Interact_249_19
PubMedID: 26915974

Title : Polyproline promotes tetramerization of recombinant human butyrylcholinesterase - Larson_2014_Biochem.J_462_329
Author(s) : Larson MA , Lockridge O , Hinrichs SH
Ref : Biochemical Journal , 462 :329 , 2014
Abstract : Human BChE (butyrylcholinesterase) protects against the toxicity of organophosphorus nerve agents and pesticides. BChE purified from human plasma is limited and pathogen carry-over is a concern. Unlike the native BChE tetrameric complex with a residence time of days, rBChE (recombinant BChE) is produced predominantly as dimers and monomers that are cleared from the circulation within minutes. Assembly into tetramers requires incorporation of proline-rich peptides, a process that was thought to occur intracellularly. Our goal was to determine whether polyproline added to rBChE under cell-free conditions would promote tetramerization. Secreted rBChE was purified by procainamide affinity chromatography, and synthetic polyprolines (8-mer to 300-mer) were tested to determine their effect on tetramer assembly. These studies demonstrated that 90-98% of purified rBChE (65 muM) could be assembled into tetramers when incubated with synthetic 17-mer or 50-mer polyproline peptides (100 muM) for 1.5 h at 25 degrees C. However, rBChE tetramerization was inefficient with smaller 8-mer polyproline peptides and larger 300-mer polyproline proteins. Collectively, these studies demonstrated that the eukaryotic cellular machinery is not required for assembly of active BChE into tetramers and that this process can occur in vitro with purified rBChE in the presence of peptides containing 15-50 consecutive proline residues.
ESTHER : Larson_2014_Biochem.J_462_329
PubMedSearch : Larson_2014_Biochem.J_462_329
PubMedID: 24916051

Title : Application of chromosomal DNA and protein targeting for the identification of Yersinia pestis - Larson_2013_Proteomics.Clin.Appl_7_416
Author(s) : Larson MA , Ding SJ , Slater SR , Hanway A , Bartling AM , Fey PD , Lockridge O , Francesconi SC , Hinrichs SH
Ref : Proteomics Clin Appl , 7 :416 , 2013
Abstract : PURPOSE: A comprehensive strategy was developed and validated for the identification of pathogens from closely related near neighbors using both chromosomal and protein biomarkers, with emphasis on distinguishing Yersinia pestis from the ancestral bacterium Yersinia pseudotuberculosis. EXPERIMENTAL DESIGN: Computational analysis was used to discover chromosomal targets unique to Y. pestis. Locus identifier YPO1670 was selected for further validation and PCR was used to confirm that this biomarker was exclusively present in Y. pestis strains, while absent in other Yersinia species. RT-PCR and Western blot analyses were utilized to evaluate YPO1670 expression and MRM MS was performed to identify the YPO1670 protein within cell lysates.
RESULTS: The described study validated that YPO1670 was exclusive to Y. pestis. PCR confirmed the locus to be unique to Y. pestis. The associated transcript and protein were produced throughout growth with the highest abundance occurring in stationary phase and MRM MS conclusively identified the YPO1670 protein in cell extracts. CONCLUSIONS AND CLINICAL RELEVANCE: These findings validated YPO1670 as a reliable candidate biomarker for Y. pestis and that a dual DNA and protein targeting approach is feasible for the development of next-generation assays to accurately differentiate pathogens from near neighbors.
ESTHER : Larson_2013_Proteomics.Clin.Appl_7_416
PubMedSearch : Larson_2013_Proteomics.Clin.Appl_7_416
PubMedID: 23436733

Title : Large direct repeats flank genomic rearrangements between a new clinical isolate of Francisella tularensis subsp. tularensis A1 and Schu S4 - Nalbantoglu_2010_PLoS.One_5_e9007
Author(s) : Nalbantoglu U , Sayood K , Dempsey MP , Iwen PC , Francesconi SC , Barabote RD , Xie G , Brettin TS , Hinrichs SH , Fey PD
Ref : PLoS ONE , 5 :e9007 , 2010
Abstract : Francisella tularensis subspecies tularensis consists of two separate populations A1 and A2. This report describes the complete genome sequence of NE061598, an F. tularensis subspecies tularensis A1 isolated in 1998 from a human with clinical disease in Nebraska, United States of America. The genome sequence was compared to Schu S4, an F. tularensis subspecies tularensis A1a strain originally isolated in Ohio in 1941. It was determined that there were 25 nucleotide polymorphisms (22 SNPs and 3 indels) between Schu S4 and NE061598; two of these polymorphisms were in potential virulence loci. Pulsed-field gel electrophoresis analysis demonstrated that NE061598 was an A1a genotype. Other differences included repeat sequences (n = 11 separate loci), four of which were contained in coding sequences, and an inversion and rearrangement probably mediated by insertion sequences and the previously identified direct repeats I, II, and III. Five new variable-number tandem repeats were identified; three of these five were unique in NE061598 compared to Schu S4. Importantly, there was no gene loss or gain identified between NE061598 and Schu S4. Interpretation of these data suggests there is significant sequence conservation and chromosomal synteny within the A1 population. Further studies are needed to determine the biological properties driving the selective pressure that maintains the chromosomal structure of this monomorphic pathogen.
ESTHER : Nalbantoglu_2010_PLoS.One_5_e9007
PubMedSearch : Nalbantoglu_2010_PLoS.One_5_e9007
PubMedID: 20140244
Gene_locus related to this paper: fratt-q5ngu5

Title : Tyrosines of human and mouse transferrin covalently labeled by organophosphorus agents: a new motif for binding to proteins that have no active site serine - Li_2009_Toxicol.Sci_107_144
Author(s) : Li B , Schopfer LM , Grigoryan H , Thompson CM , Hinrichs SH , Masson P , Lockridge O
Ref : Toxicol Sci , 107 :144 , 2009
Abstract : The expectation from the literature is that organophosphorus (OP) agents bind to proteins that have an active site serine. However, transferrin, a protein with no active site serine, was covalently modified in vitro by 0.5mM 10-fluoroethoxyphosphinyl-N-biotinamido pentyldecanamide, chlorpyrifos oxon, diisopropylfluorophosphate, dichlorvos, sarin, and soman. The site of covalent attachment was identified by analyzing tryptic peptides in the mass spectrometer. Tyr 238 and Tyr 574 in human transferrin and Tyr 238, Tyr 319, Tyr 429, Tyr 491, and Tyr 518 in mouse transferrin were labeled by OP. Tyrosine in the small synthetic peptide ArgTyrThrArg made a covalent bond with diisopropylfluorophosphate, chlorpyrifos oxon, and dichlorvos at pH 8.3. These results, together with our previous demonstration that albumin and tubulin bind OP on tyrosine, lead to the conclusion that OP bind covalently to tyrosine, and that OP binding to tyrosine is a new OP-binding residue. The OP-reactive tyrosines are activated by interaction with Arg or Lys. It is suggested that many proteins in addition to those already identified may be modified by OP on tyrosine. The extent to which tyrosine modification by OP can occur in vivo and the toxicological implications of such modifications require further investigation.
ESTHER : Li_2009_Toxicol.Sci_107_144
PubMedSearch : Li_2009_Toxicol.Sci_107_144
PubMedID: 18930948

Title : Complete genome sequence of Francisella tularensis subspecies holarctica FTNF002-00 - Barabote_2009_PLoS.One_4_e7041
Author(s) : Barabote RD , Xie G , Brettin TS , Hinrichs SH , Fey PD , Jay JJ , Engle JL , Godbole SD , Noronha JM , Scheuermann RH , Zhou LW , Lion C , Dempsey MP
Ref : PLoS ONE , 4 :e7041 , 2009
Abstract : Francisella tularensis subspecies holarctica FTNF002-00 strain was originally obtained from the first known clinical case of bacteremic F. tularensis pneumonia in Southern Europe isolated from an immunocompetent individual. The FTNF002-00 complete genome contains the RD(23) deletion and represents a type strain for a clonal population from the first epidemic tularemia outbreak in Spain between 1997-1998. Here, we present the complete sequence analysis of the FTNF002-00 genome. The complete genome sequence of FTNF002-00 revealed several large as well as small genomic differences with respect to two other published complete genome sequences of F. tularensis subsp. holarctica strains, LVS and OSU18. The FTNF002-00 genome shares >99.9% sequence similarity with LVS and OSU18, and is also approximately 5 MB smaller by comparison. The overall organization of the FTNF002-00 genome is remarkably identical to those of LVS and OSU18, except for a single 3.9 kb inversion in FTNF002-00. Twelve regions of difference ranging from 0.1-1.5 kb and forty-two small insertions and deletions were identified in a comparative analysis of FTNF002-00, LVS, and OSU18 genomes. Two small deletions appear to inactivate two genes in FTNF002-00 causing them to become pseudogenes; the intact genes encode a protein of unknown function and a drug:H(+) antiporter. In addition, we identified ninety-nine proteins in FTNF002-00 containing amino acid mutations compared to LVS and OSU18. Several non-conserved amino acid replacements were identified, one of which occurs in the virulence-associated intracellular growth locus subunit D protein. Many of these changes in FTNF002-00 are likely the consequence of direct selection that increases the fitness of this subsp. holarctica clone within its endemic population. Our complete genome sequence analyses lay the foundation for experimental testing of these possibilities.
ESTHER : Barabote_2009_PLoS.One_4_e7041
PubMedSearch : Barabote_2009_PLoS.One_4_e7041
PubMedID: 19756146
Gene_locus related to this paper: fratt-q5ngu5

Title : Pseudo-esterase activity of human albumin: slow turnover on tyrosine 411 and stable acetylation of 82 residues including 59 lysines - Lockridge_2008_J.Biol.Chem_283_22582
Author(s) : Lockridge O , Xue W , Gaydess A , Grigoryan H , Ding SJ , Schopfer LM , Hinrichs SH , Masson P
Ref : Journal of Biological Chemistry , 283 :22582 , 2008
Abstract : Human albumin is thought to hydrolyze esters because multiple equivalents of product are formed for each equivalent of albumin. Esterase activity with p-nitrophenyl acetate has been attributed to turnover at tyrosine 411. However, p-nitrophenyl acetate creates multiple, stable, acetylated adducts, a property contrary to turnover. Our goal was to identify residues that become acetylated by p-nitrophenyl acetate and determine the relationship between stable adduct formation and turnover. Fatty acid-free human albumin was treated with 0.5 mm p-nitrophenyl acetate for 5 min to 2 weeks, or with 10 mm p-nitrophenyl acetate for 48 h to 2 weeks. Aliquots were digested with pepsin, trypsin, or GluC and analyzed by mass spectrometry to identify labeled residues. Only Tyr-411 was acetylated within the first 5 min of reaction with 0.5 mm p-nitrophenyl acetate. After 0.5-6 h there was partial acetylation of 16-17 residues including Asp-1, Lys-4, Lys-12, Tyr-411, Lys-413, and Lys-414. Treatment with 10 mm p-nitrophenyl acetate resulted in acetylation of 59 lysines, 10 serines, 8 threonines, 4 tyrosines, and Asp-1. When Tyr-411 was blocked with diisopropylfluorophosphate or chlorpyrifos oxon, albumin had normal esterase activity with beta-naphthyl acetate as visualized on a nondenaturing gel. However, after 82 residues had been acetylated, esterase activity was almost completely inhibited. The half-life for deacetylation of Tyr-411 at pH 8.0, 22 degrees C was 61 +/- 4 h. Acetylated lysines formed adducts that were even more stable. In conclusion, the pseudo-esterase activity of albumin is the result of irreversible acetylation of 82 residues and is not the result of turnover.
ESTHER : Lockridge_2008_J.Biol.Chem_283_22582
PubMedSearch : Lockridge_2008_J.Biol.Chem_283_22582
PubMedID: 18577514

Title : Increased hepatotoxicity and cardiac fibrosis in cocaine-treated butyrylcholinesterase knockout mice - Duysen_2008_Basic.Clin.Pharmacol.Toxicol_103_514
Author(s) : Duysen EG , Li B , Carlson M , Li YF , Wieseler S , Hinrichs SH , Lockridge O
Ref : Basic Clin Pharmacol Toxicol , 103 :514 , 2008
Abstract : In mice, cocaine is detoxified to inactive products by butyrylcholinesterase (BChE) and carboxylesterase. In human beings, cocaine detoxification is primarily by BChE. The focus of this investigation was to elucidate the importance of BChE in reducing pathophysiological effects following cocaine exposure. Previous studies examining the effects of cocaine on BChE deficient animals relied on chemical inhibition of BChE with tetraisopropyl pyrophosphoramide (iso-OMPA). The creation of the BChE knockout mouse has provided a model for studying pathological effects of cocaine in mice free of chemical confounders. We hypothesized that mice with low or no BChE activity would have reduced cocaine metabolism, leading to hepatotoxicity and cardiomyopathy. A high-resolution in vivo imaging system recorded cardiac and respiratory function following treatment with a carboxylesterase inhibitor and a high dose of cocaine (100 mg/kg, intraperitoneally). The BChE-/- mice demonstrated depressed respiration through 12 hr after dosing and abnormal respiratory patterns consisting of a pause at full inspiration (apneusis), whereas BChE+/+ mice had recovered normal respiration rates by 30 min. after dosing and exhibited no apneusis. Liver and cardiac histology sections were analysed following a 20 mg/kg intraperitoneally dose of cocaine administered daily for 7 days. BChE-/- mice treated for 7 days with the chronic low dose showed significant hepatotoxicity and cardiac perivascular fibrosis compared to BChE+/+ mice. The observed functional changes following acute high-dose and chronic low-dose cocaine in BChE-/- and +/- mice warrants further investigation into the possibility of increased cocaine toxicity in human beings with BChE deficiency.
ESTHER : Duysen_2008_Basic.Clin.Pharmacol.Toxicol_103_514
PubMedSearch : Duysen_2008_Basic.Clin.Pharmacol.Toxicol_103_514
PubMedID: 19067679

Title : Five tyrosines and two serines in human albumin are labeled by the organophosphorus agent FP-biotin - Ding_2008_Chem.Res.Toxicol_21_1787
Author(s) : Ding SJ , Carr J , Carlson JE , Tong L , Xue W , Li Y , Schopfer LM , Li B , Nachon F , Asojo OA , Thompson CM , Hinrichs SH , Masson P , Lockridge O
Ref : Chemical Research in Toxicology , 21 :1787 , 2008
Abstract : Tyrosine 411 of human albumin is an established site for covalent attachment of 10-fluoroethoxyphosphinyl- N-biotinamidopentyldecanamide (FP-biotin), diisopropylfluorophosphate, chlorpyrifos oxon, soman, sarin, and dichlorvos. This work investigated the hypothesis that other residues in albumin could be modified by organophosphorus agents (OP). Human plasma was aggressively treated with FP-biotin; plasma proteins were separated into high and low abundant portions using a proteome partitioning antibody kit, and the proteins were digested with trypsin. The FP-biotinylated tryptic peptides were isolated by binding to monomeric avidin beads. The major sites of covalent attachment identified by mass spectrometry were Y138, Y148, Y401, Y411, Y452, S232, and S287 of human albumin. Prolonged treatment of pure human albumin with chlorpyrifos oxon labeled Y138, Y150, Y161, Y401, Y411, and Y452. To identify the most reactive residue, albumin was treated for 2 h with DFP, FP-biotin, chlorpyrifos oxon, or soman, digested with trypsin or pepsin, and analyzed by mass spectrometry. The most reactive residue was always Tyr 411. Diethoxyphosphate-labeled Tyr 411 was stable for months at pH 7.4. These results will be useful in the development of specific antibodies to detect OP exposure and to engineer albumin for use as an OP scavenger.
ESTHER : Ding_2008_Chem.Res.Toxicol_21_1787
PubMedSearch : Ding_2008_Chem.Res.Toxicol_21_1787
PubMedID: 18707141

Title : Matrix-assisted laser desorption\/ionization time-of-flight mass spectrometry assay for organophosphorus toxicants bound to human albumin at Tyr411 - Li_2007_Anal.Biochem_361_263
Author(s) : Li B , Schopfer LM , Hinrichs SH , Masson P , Lockridge O
Ref : Analytical Biochemistry , 361 :263 , 2007
Abstract : Our goal was to determine whether chlorpyrifos oxon, dichlorvos, diisopropylfluorophosphate (DFP), and sarin covalently bind to human albumin. Human albumin or plasma was treated with organophosphorus (OP) agent at alkaline pH, digested with pepsin at pH 2.3, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Two singly charged peaks m/z 1718 and 1831, corresponding to the unlabeled peptide fragments containing the active site Tyr411 residue, were detected in all samples. The sequences of the two peptides were VRYTKKVPQVSTPTL and LVRYTKKVPQVSTPTL. The peptide-OP adducts of these peptides were also found. They had masses of 1854 and 1967 for chlorpyrifos oxon, 1825 and 1938 for dichlorvos, 1881 and 1994 for DFP, and 1838 and 1938 for sarin; these masses fit a mechanism whereby OP bound covalently to Tyr411. The binding of DFP to Tyr411 of human albumin was confirmed by electrospray tandem mass spectrometry and analysis of product ions. None of the OP-albumin adducts lost an alkoxy group, leading to the conclusion that aging did not occur. Our results show that OP pesticides and nerve agents bind covalently to human albumin at Tyr411. The presence of Tyr411 on an exposed surface of albumin suggests that an antibody response could be generated against OP-albumin adducts.
ESTHER : Li_2007_Anal.Biochem_361_263
PubMedSearch : Li_2007_Anal.Biochem_361_263
PubMedID: 17188226

Title : Phenotype of the adult acetylcholinesterase-deficient mouse. -
Author(s) : Duysen EG , Stribley JA , Fry DL , Hinrichs SH , Lockridge O
Ref : Cholinergic Mechanisms, CRC Press :559 , 2004
PubMedID:

Title : Rescue of the acetylcholinesterase knockout mouse by feeding a liquid diet\; phenotype of the adult acetylcholinesterase deficient mouse - Duysen_2002_Brain.Res.Dev.Brain.Res_137_43
Author(s) : Duysen EG , Stribley JA , Fry DL , Hinrichs SH , Lockridge O
Ref : Brain Research Developmental Brain Research , 137 :43 , 2002
Abstract : Acetylcholinesterase (AChE, EC3.1.1.7) functions in nerve impulse transmission, and possibly as a cell adhesion factor during neurite outgrowth. These functions predicted that a mouse with zero AChE activity would be unable to live. It was a surprise to find that AChE -/- mice were born alive and survived an average of 14 days. The emaciated appearance of AChE -/- mice suggested an inability to obtain sufficient nutrition and experiments were undertaken to increase caloric intake. Pregnant and lactating dams (+/-) were fed 11% high fat chow supplemented with liquid Ensure. AChE -/- pups were weaned early, on day 15, and fed liquid Ensure. Although nullizygous animals showed slow but steady weight gain with survival over 1 year (average 100 days), they remained small at all ages compared to littermates. They demonstrated delays in temperature regulation (day 22 vs. 15), eye opening (day 13 vs. 12), righting reflex (day 18 vs. 12), descent of testes (week 7-8 vs. 4), and estrous (week 15-16 vs. 6-7). Significant physical findings in adult AChE -/- mice included body tremors, abnormal gait and posture, absent grip strength, inability to eat solid food, pinpoint pupils, decreased pain response, vocalization, and early death caused by seizures or gastrointestinal tract ileus. Behavioral deficits included urination and defecation in the nest, lack of aggression, reduced pain perception, and sexual dysfunction. These findings support the classical role for AChE in nerve impulse conduction and further suggest that AChE is essential for timely physical development and higher brain function.
ESTHER : Duysen_2002_Brain.Res.Dev.Brain.Res_137_43
PubMedSearch : Duysen_2002_Brain.Res.Dev.Brain.Res_137_43
PubMedID: 12128253

Title : Abundant tissue butyrylcholinesterase and its possible function in the acetylcholinesterase knockout mouse - Li_2000_J.Neurochem_75_1320
Author(s) : Li B , Stribley JA , Ticu A , Xie W , Schopfer LM , Hammond P , Brimijoin S , Hinrichs SH , Lockridge O
Ref : Journal of Neurochemistry , 75 :1320 , 2000
Abstract : We have described recently an acetylcholinesterase (AChE) knockout mouse. While comparing the tissue distribution of AChE and butyrylcholinesterase (BChE), we found that extraction buffers containing Triton X-100 strongly inhibited mouse BChE activity. In contrast, buffers with Tween 20 caused no inhibition of BChE. Conventional techniques grossly underestimated BChE activity by up to 15-fold. In Tween 20 buffer, the intestine, serum, lung, liver, and heart had higher BChE than AChE activity. Only brain had higher AChE than BChE activity in AChE +/+ mice. These findings contradict the dogma, based mainly on observations in Triton X-100 extracts, that BChE is a minor cholinesterase in animal tissues. AChE +/- mice had 50% of normal AChE activity and AChE -/- mice had none, but all mice had similar levels of BChE activity. BChE was inhibited by Triton X-100 in all species tested, except rat and chicken. Inhibition was reversible and competitive with substrate binding. The active site of rat BChE was unique, having an arginine in place of leucine at position 286 (human BChE numbering) in the acyl-binding pocket of the active site, thus explaining the lack of inhibition of rat BChE by Triton X-100. The generally high levels of BChE activity in tissues, including the motor endplate, and the observation that mice live without AChE, suggest that BChE has an essential function in nullizygous mice and probably in wild-type mice as well.
ESTHER : Li_2000_J.Neurochem_75_1320
PubMedSearch : Li_2000_J.Neurochem_75_1320
PubMedID: 10936216

Title : Postnatal developmental delay and supersensitivity to organophosphate in gene-targeted mice lacking acetylcholinesterase - Xie_2000_J.Pharmacol.Exp.Ther_293_896
Author(s) : Xie W , Stribley JA , Chatonnet A , Wilder PJ , Rizzino A , McComb RD , Taylor P , Hinrichs SH , Lockridge O
Ref : Journal of Pharmacology & Experimental Therapeutics , 293 :896 , 2000
Abstract : Acetylcholinesterase (AChE; EC 3.1.1.7) is the primary terminator of nerve impulse transmission at cholinergic synapses and is believed to play an important role in neural development. Targeted deletion of four exons of the ACHE gene reduced AChE activity by half in heterozygous mutant mice and totally eliminated AChE activity in nullizygous animals. Butyrylcholinesterase (EC 3.1.1.8) activity was normal in AChE -/- mice. Although nullizygous mice were born alive and lived up to 21 days, physical development was delayed. The neuromuscular junction of 12-day-old nullizygous animals appeared normal in structure. Nullizygous mice were highly sensitive to the toxic effects of the organophosphate diisopropylfluorophosphate and to the butyrylcholinesterase-specific inhibitor bambuterol. These findings indicate that butyrylcholinesterase and possibly other enzymes are capable of compensating for some functions of AChE and that the inhibition of targets other than AChE by organophosphorus agents results in death.
ESTHER : Xie_2000_J.Pharmacol.Exp.Ther_293_896
PubMedSearch : Xie_2000_J.Pharmacol.Exp.Ther_293_896
PubMedID: 10869390
Gene_locus related to this paper: mouse-ACHE

Title : Knockout of one acetylcholinesterase allele in the mouse - Xie_1999_Chem.Biol.Interact_119-120_289
Author(s) : Xie W , Wilder PJ , Stribley J , Chatonnet A , Rizzino A , Taylor P , Hinrichs SH , Lockridge O
Ref : Chemico-Biological Interactions , 119-120 :289 , 1999
Abstract : One allele of the AChE gene (ACHE) was knocked out in embryonic stem (ES) cells by homologous recombination. The targeting vector contained 2 kb of a TK gene cassette for negative selection, 884 bp of ACHE including exon 1, 1.6 kb of a Neo(r) gene cassette for positive selection, 5.2 kb of the ACHE Bam HI fragment including exon 6, and 3 kb of Bluescript. The use of this vector deleted exons 2-5, which removed 93% of the ACHE coding sequence including the signal peptide, the active site serine, and the histidine and glutamic acid of the catalytic triad. The gene targeting vector was transfected into ES cells by electroporation. Colonies resistant to G418 and gancyclovir were screened for homologous recombination by Southern blotting. Out of 200 colonies, four were found to have undergone homologous recombination. These four ACHE (+/-) ES cell lines were expanded to provide cells for microinjection into C57Bl/6 mouse blastocysts. The injected blastocysts were implanted into pseudopregnant CD/l white mice. More than 200 injected blastocysts were transferred into 20 mice. More than 65 mice were born, of which 11 were chimeras. Chimeras were identified by their black and agouti coat color. Littermates were all black. Thus far, seven male chimeras have been bred with more than 130 C57Bl/6 females to generate 26 agouti mice out of 199 living offspring. This demonstrated that the ACHE (+/-) ES cells contributed to the germline. Offspring with agouti coat color have a 50% chance of carrying the knockout allele. The 26 agouti offspring were screened for an ACHE (+/-) genotype by tail biopsy PCR. Ten out of 26 agouti mice are heterozygous ACHE knockout mice, and they are healthy and alive at 29 days of age. We expect a phenotype to appear in nullizygous animals.
ESTHER : Xie_1999_Chem.Biol.Interact_119-120_289
PubMedSearch : Xie_1999_Chem.Biol.Interact_119-120_289
PubMedID: 10421464