Purification by Affinity Chromatography of Red Cell Membrane Acetylcholinesterase

Brodbeck, Urs; Gentinetta, R.; Ott, Peter

doi:10.1007/978-3-642-68077-9_9

Cited by 31 publications

(9 citation statements)

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“…The specific enzymatic activity obtained for the extract of solubilized membranes of HUVECs was of 13.0 UI·mg protein −1 . This value is greater than those obtained in human cerebrospinal fluid (≈ 4 UI·mg −1 [14]); in human ocular fluid (≈ 0.04 UI·mg −1 [15,16]), but lower than the results obtained for the AChE purified from human erythrocytes (582 UI·mg −1 of AChE [17]).…”

Section: Discussioncontrasting

confidence: 54%

“…The specific enzymatic activity obtained for the extract of solubilized membranes of HUVECs was of 13.0 UIAEmg protein )1 . This value is greater than those obtained in human cerebrospinal fluid ( 4 UIAEmg )1 [14]); in human ocular fluid ( 0.04 UIAEmg )1 [15,16]), but lower than the results obtained for the AChE purified from human erythrocytes (582 UIAEmg )1 of AChE [17]). From the literature, we could expect this membrane isolation and solubilization procedure to be inadequate for measurements of AChE activity or for determination of protein concentration.…”

Section: Discussionmentioning

confidence: 57%

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Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

Carvalho¹,

Graça²,

Martins‐Silva³

et al. 2005

The FEBS Journal

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Acetylcholinesterase is an enzyme whose best‐known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X‐100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X‐100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C‐terminal anti‐acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N‐terminal anti‐acetylcholinesterase IgG antibody revealed a double band around 66–70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane‐bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.

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Section: Discussioncontrasting

confidence: 54%

Section: Discussionmentioning

confidence: 57%

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

Carvalho¹,

Graça²,

Martins‐Silva³

et al. 2005

The FEBS Journal

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“…DS-AChE and proteinase K solubifized AChE from Torpedo marmorata were purified according to [8] and DS-AChE from human erythrocyte membranes as described in [9]. AChE activity was monitored according to Ellman et al [IO], membrane form VSG (ILTat 1.25) was prepared as described 171; phospholipase C type III from B. cereus was from Sigma.…”

Section: Methodsmentioning

confidence: 99%

The membrane‐anchoring systems of vertebrate acetylcholinesterase and variant surface glycoproteins of African trypanosomes share a common antigenic determinant

et al. 1986

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Amphiphilic detergent-soluble acetylcholinesterase (AChE) from Torpedo is converted to a hydrophilic form by digestion with phospholipase C from Trypanosoma brucei or from Bacillus cereus. This lipase digestion uncovers an immunological determinant which crossreacts with a complex carbohydrate structure present in the hydrophilic form of all variant surface glycoproteins (VSG) of T. brucei. This crossreacting determinant is also detected in human erythrocyte AChE after digestion with T. brucei lipase. From these results we conclude that the glycophospholipid anchors of protozoan VSG and of AChE of the two vertebrates share common structuraf features, suggesting that this novel type of membrane anchor has been conserved during evolution. Acetylchohnesterase Phospholipase C Surface glycoprotein (Trypanosoma brucei)Crossreacting determinant

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“…The enzyme was solubilized with 10 mM Na-phosphate buffer (pH 7.4) which contained 1% Triton X-100 and 0.05% NAN3. The solubilized acetylcholinesterase was purified by 2 subsequent affinity chromatography steps, essentially as in [6]. Triton X-100 was removed from the enzyme preparation by chromatography on hydroxylapatite (Bio-Gel HTP), and the protein eluted from the column with a 20 mM sodium phosphate buffer (pH 7.4) containing 0.1 M NaCI, 3 mM NAN3, 0.2 mM EDTA and 1% octyl-tetraoxyethylene.…”

Section: Methodsmentioning

confidence: 99%

Acetylcholinesterase from human erythrocyte membranes: dimers as functional units

et al. 1982

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Purification by Affinity Chromatography of Red Cell Membrane Acetylcholinesterase

Cited by 31 publications

References 9 publications

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

The membrane‐anchoring systems of vertebrate acetylcholinesterase and variant surface glycoproteins of African trypanosomes share a common antigenic determinant

Acetylcholinesterase from human erythrocyte membranes: dimers as functional units

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