Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes

Nieto, Susana; Campoy, Francisco J.; Muñoz-Delgado, Encarnación; Vidal, Cecilio J.

doi:10.1016/s1357-2725(03)00007-4

Cited by 19 publications

(19 citation statements)

References 40 publications

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“…Although the low sensitivity of kidney (Fig. 1B) and erythrocyte acetylcholinesterase to PIPLC [38] might suggest a blood origin for renal acetylcholinesterase, the presence in kidney of G 1 A acetylcholinesterase, which is absent from erythrocytes, and the difference between acetylcholinesterases of kidney and erythrocytes in the extent of binding with the lectins concanavalin A, Lens culinaris agglutinin (LCA), and Ricinus communis agglutinin (RCA) (Fig. 3), ruled out the blood origin and supported the renal cells themselves as the most probable source of kidney acetylcholinesterase.…”

Section: Resultsmentioning

confidence: 99%

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Muñoz-Delgado¹,

Montenegro²,

Campoy³

et al. 2010

The FEBS Journal

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Despite the aberrant expression of cholinesterases in tumours, the question of their possible contribution to tumorigenesis remains unsolved. The identification in kidney of a cholinergic system has paved the way to functional studies, but details on renal cholinesterases are still lacking. To fill the gap and to determine whether cholinesterases are abnormally expressed in renal tumours, paired pieces of normal kidney and renal cell carcinomas (RCCs) were compared for cholinesterase activity and mRNA levels. In studies with papillary RCC (pRCC), conventional RCC, chromophobe RCC, and renal oncocytoma, acetylcholinesterase activity increased in pRCC (3.92 ± 3.01 mUAEmg )1 , P = 0.031) and conventional RCC (2.64 ± 1.49 mUAEmg , P = 0.031). Glycosylphosphatidylinositollinked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase tetramers predominated in control and cancerous kidney. Acetylcholinesterase mRNAs with exons E1c and E1e, 3¢-alternative T, H and R acetylcholinesterase mRNAs and butyrylcholinesterase mRNA were identified in kidney. The levels of acetylcholinesterase and butyrylcholinesterase mRNAs were nearly 1000-fold lower in human kidney than in colon. Whereas kidney and renal tumours showed comparable levels of acetylcholinesterase mRNA, the content of butyrylcholinesterase mRNA was increased 10-fold in pRCC. The presence of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, and the prevalence of glycosylphosphatidylinositol-anchored acetylcholinesterase explains the splicing to acetylcholinesterase-H mRNA. The consequences of butyrylcholinesterase upregulation for pRCC growth are discussed.

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Section: Resultsmentioning

confidence: 99%

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Muñoz-Delgado¹,

Montenegro²,

Campoy³

et al. 2010

The FEBS Journal

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“…The asymmetric structure of 16.2S AChE was assessed by cleaving its ColQ tail with collagenase [20]. The dimeric state of the 4.4S species (in gradients with Brij 96) and the monomeric state of the 3.2S components were assessed by the conversion of the former into the latter variants by reducing the disulfide bond which tethers the subunits in dimers [21]. The linkage of GPI to G 2 A AChE was tested by its exposure to phosphatidylinositol-specific phospholipase C (PIPLC) from Bacillus thuringiensis.…”

Section: Methodsmentioning

confidence: 99%

“…PIPLC alone failed to convert gut G 2 A into G 2 H AChE, which indicated the presence of acyl chains linked to the inositol ring. Taking advantage of the capacity of alkaline hydroxylamine for removing the acyl groups, the conversion of G 2 A into G 2 H forms was attempted by subjecting the sample to hydroxylaminolysis before the PIPLC treatment [21]. Research Article 2177 synthesis of cDNA was carried out for 50 min at 42 °C, in a volume of 20 μl.…”

Section: Methodsmentioning

confidence: 99%

Cholinesterases are down-expressed in human colorectal carcinoma

Montenegro

Ruíz-Espejo

Campoy

et al. 2006

Cell. Mol. Life Sci.

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The aberrations of cholinesterase (ChE) genes and the variation of ChE activity in cancerous tissues prompted us to investigate the expression of ChEs in colorectal carcinoma. The study of 55 paired specimens of healthy (HG) and cancerous gut (CG) showed that acetylcholinesterase (AChE) activity fell by 32% and butyrylcholinesterase (BuChE) activity by 58% in CG. Abundant AChE-H, fewer AChE-T, and even fewer AChE-R and BuChE mRNAs were observed in HG, and their content was greatly diminished in CG. The high level of the AChE-H mRNA explains the abundance of AChE-H subunits in HG, which as glycosylphosphatidylinositol (GPI)-anchored amphiphilic AChE dimers (G2(A)) and monomers (G1(A)) account for 69% of AChE activity. The identification of AChE-T and BuChE mRNAs justifies the occurrence in gut of A12, G4(H) and PRiMA-containing G4(A) AChE forms, besides G4(H), G4(A) and G1(H) BuChE. The down-regulation of ChEs might contribute to gut carcinogenesis by increasing acetylcholine availability and over-stimulating muscarinic receptors.

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“…Using SDS/PAGE electrophoresis in the presence of dithiothreitol and 2‐mercaptoethanol, and western blotting with a specific anti‐AChE Ig for the C‐terminal region, a single protein band was observed of approximately 70 kDa. This molecular mass is the expected size for the human monomeric AChE in other cell types (human erythrocytes [21], human blood lymphocytes [22], mouse erythrocytes [23] and cotton aphid [24]). With a specific anti‐AChE Ig for the N‐terminal region, a double band around 66–70 kDa corresponding of two monomeric distinct forms of AChE was observed.…”

Section: Discussionmentioning

confidence: 99%

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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Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes

Cited by 19 publications

References 40 publications

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Cholinesterases are down-expressed in human colorectal carcinoma

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

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