Human erythrocyte acetylcholinesterase purification, properties and kinetic behavior

Ciliv, G; Ozand, P

doi:10.1016/0005-2744(72)90053-8

Cited by 35 publications

(5 citation statements)

References 39 publications

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“…The finding is not surprising because it is known from literature [21][22][23] . The K m and V max values are in a good correspondence with literature as well [24][25][26] . Saturation curve for 2,6-dichlorophenolindophenol acetate is depicted as Figure 4.…”

Section: Resultssupporting

confidence: 74%

Evaluation of 2,6-dichlorophenolindophenol acetate as a substrate for acetylcholinesterase activity assay

Pohanka

Holas

2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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Ellman's method is a standard protocol for the determination of cholinesterases activity. Though the method is ready for laboratory purposes, it has some drawbacks as well. In the current article, 2,6-dichloroindophenol acetate is performed as a chromogenic substrate suitable for acetylcholinesterase (AChE) activity examination. Michaelis constant and maximal velocity for 2,6-dichloroindophenol acetate were determined (38.0 mM and 244 pkat) and compared to the values for acetythiocholine (K m 0.18 mM; V max 5.1 nkat). Docking for 2,6-dichloroindophenol acetate and human AChE was done as well. In conclusion, 2,6-dichloroindophenol acetate seems to be suitable chromogenic substrate for AChE and spectrophotometry and based on this it can be easily performed whenever AChE activity should be tested.

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Section: Resultssupporting

confidence: 74%

Evaluation of 2,6-dichlorophenolindophenol acetate as a substrate for acetylcholinesterase activity assay

Pohanka

Holas

2015

Journal of Enzyme Inhibition and Medicinal Chemistry

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“…pulcherrimus embryos also tended to form aggregates in the absence of Triton X-100, Triton X-100 was added to the elution buffer for analytical gel filtration. It has also been reported that the purified AchE from human erythrocyte easily forms reversible aggregates in deionized water (18). Although it is difficult to make a strict estimation of the molecular weight of AchE in the presence of Triton X-100, the molecular weight of Hemicentrotus AchE was roughly estimated to be 3 x lo5, and the value was comparalbe with that of Pseudocentrotus depressus (2).…”

Section: Discussionmentioning

confidence: 95%

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

et al. 1986

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Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage.Purification of AchE from plutei has been carried out by the procedure including affinity chromatography. Purified AchE had the activity 14,600 times higher than that of homogenate, and the final yield of AchE was 8%. The enzyme seems to be electrophoretically homogeneous, and has a molecular weight of 3 x lo5 as determined by Sepharose CL6B column chromatography.In sea urchin embryos, AchE activity is little detectable before gastrula stage, and at this stage AchE activity begins to increase sharply (1, 2). However, it is not known whether the sudden appearance of this enzyme is due to the onset of transcription of AchE gene or due to the onset of translation of the maternal stored mRNA of AchE. WHITTAKER (3) and SATOH and IKEGAMI (4, 5) presented the evidences suggesting that acidian embryos express the AchE gene activity at the certain time of development and that the embryonic cells seems to determine the timing of the expression of this gene seems by somehow measuring the number of DNA replication cycles.As the first step for the elucidation of the molecular mechanism underlying the production of this enzyme by gastrula cells, we attempted to purify AchE from sea urchin embryos. Previously the partial characterization of sea urchin AchE has been reported by others using the partially purified enzyme preparation (2). In the present paper, we describe a procedure for the preparation of AchE from sea urchin embryos. The procedure contains the affinity chromatography on a trimethyl (p-aminophenyl) ammonium-Sepharose (TMAPA-Sepharose) column developed by BERMAN and YOUNG (6) for the purification of AchE from eel electric tissue and bovine erythrocytes. MATEFUALS AND METHODS Preparation of embryoEggs and sperm of Hemicentrotus pulcherrimus were collected by artificial spawning induced by a KCl solution. Inseminated eggs were raised in filtered sea water at 20°C with a constant stirring. Embryos were harvested by centrifugation, and kept frozen at -20°C until use. Enzyme assayThe activity of AchE was assayed by the method of Ellman et al. (7), using 2.3 mM acetylthiocholine iodide as a substrate.For the determination of ontogenic change of the enzyme activity, los embryos were suspended in 5 ml of ice-cold deionized water and sonicated by a Branson Sonifier at 40 W for 30 sec. An aliquot of the sonicate was 85

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“…Because DFP and BNPP are organophosphates, general CES inhibitors, it is plausible that DFP also inhibits AADAC. Eserine and NaF are cholinesterase inhibitors (Iwatsubo, 1965;Ciliv and Ozand, 1972;Preuss and Svensson, 1996). It is of interest that flutamide hydrolysis was effectively inhibited by eserine but not by NaF.…”

Section: Discussionmentioning

confidence: 99%

Human Arylacetamide Deacetylase Is a Principal Enzyme in Flutamide Hydrolysis

Watanabe

Fukami²,

Nakajima³

et al. 2009

Drug Metab Dispos

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ABSTRACT:Flutamide, an antiandrogen drug, is widely used for the treatment of prostate cancer. The initial metabolic pathways of flutamide are hydroxylation and hydrolysis. It was recently reported that the hydrolyzed product, 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1), is further metabolized to N-hydroxy FLU-1, an assumed hepatotoxicant. However, the esterase responsible for the flutamide hydrolysis has not been characterized. In the present study, we found that human arylacetamide deacetylase (AADAC) efficiently hydrolyzed flutamide using recombinant AADAC expressed in COS7 cells. In contrast, carboxylesterase1 (CES1) and CES2, which are responsible for the hydrolysis of many drugs, could not hydrolyze flutamide. AADAC is specifically expressed in the endoplasmic reticulum. Flutamide hydrolase activity was highly detected in human liver microsomes (K m , 794 ؎ 83 M; V max , 1.1 ؎ 0.0 nmol/min/mg protein), whereas the activity was extremely low in human liver cytosol. The flutamide hydrolase activity in human liver microsomes was strongly inhibited by bis-(nonylphenyl)-phenylphosphate, diisopropylphosphorofluoride, and physostigmine sulfate (eserine) but moderately inhibited by sodium fluoride, phenylmethylsulfonyl fluoride, and disulfiram. The same inhibition pattern was obtained with the recombinant AADAC. Moreover, human liver and jejunum microsomes showing AADAC expression could hydrolyze flutamide, but human pulmonary and renal microsomes, which do not express AADAC, showed slight activity. In human liver microsomal samples (n ‫؍‬ 50), the flutamide hydrolase activities were significantly correlated with the expression levels of AADAC protein (r ‫؍‬ 0.66, p < 0.001). In conclusion, these results clearly showed that flutamide is exclusively hydrolyzed by AADAC. AADAC would be an important enzyme responsible for flutamideinduced hepatotoxicity.Flutamide (3Ј-trifluoro-2-methyl-4Ј-nitro-2-methyl-propinoylanilide) is a nonsteroidal antiandrogen drug used for the treatment of prostate cancer. The combination of luteinizing hormone-releasing hormone agonist results in prolonged survival in prostate cancer patients (Crawford et al., 1989). However, flutamide occasionally causes severe hepatotoxicity (Thole et al., 2004). Flutamide itself is not toxic when used at the appropriate clinical dose, but bioactivation of flutamide has been considered to be the cause of flutamide-induced hepatotoxicity (Fau et al., 1994).Flutamide is mainly metabolized to 2-hydroxyflutamide by human CYP1A2. It has been suggested that 2-hydroxyflutamide is associated with the therapeutic effect of flutamide (Katchen and Buxbaum, 1975). Flutamide is also hydrolyzed to 4-nitro-3-(trifluoromethyl)phenylamine (FLU-1) by esterase (Katchen and Buxbaum, 1975;Schulz et al., 1988). FLU-1 is considered to have no therapeutic effect (Aizawa et al., 2003). Goda et al. (2006) recently reported that FLU-1 is further metabolized to N-hydroxyl FLU-1 by human CYP3A4. Many researchers have reported on the relationship between the toxicity and metabolism...

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Human erythrocyte acetylcholinesterase purification, properties and kinetic behavior

Cited by 35 publications

References 39 publications

Evaluation of 2,6-dichlorophenolindophenol acetate as a substrate for acetylcholinesterase activity assay

Evaluation of 2,6-dichlorophenolindophenol acetate as a substrate for acetylcholinesterase activity assay

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

Human Arylacetamide Deacetylase Is a Principal Enzyme in Flutamide Hydrolysis

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