Amphiphilic and hydrophilic forms of acetyl‐ and butyrylcholinesterase in human brain

Abstract

Human brain acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) were sequentially extracted, first with a Tris-saline buffer (S1) and then with 1% (w/v) Triton X-100 (S2). About 20 and 30% of the AChE and BuChE activities were recovered in S1 and most of the remaining enzymes in S2. Main molecular forms of about 10.5 S and 12.0 S, G4 forms of AChE and BuChE, and smaller amounts of 4.5 S and 5.5 S forms, G1 species of AChE and BuChE, were measured in S1. Application of Triton X-114 phase partitioning and affinity chromatography on phenyl-agarose to S1 revealed that 25% of the AChE and none of the BuChE molecules displayed amphiphilic properties. Analysis of the enzyme activity retained by the phenyl-agarose showed that G1 AChE constituted the bulk of the amphiphilic molecules released without detergent. Main G4 forms of AChE and BuChE were found in the S2 extract. Eighty and 45% of the AChE and BuChE activities in S2 were measured in the detergent-rich phase by Triton X-114 phase partitioning. Thus, most of the AChE and about half of the BuChE molecules in S2 displayed amphiphilic properties. The main peak of BuChE, a 12.0 S form in gradients made with Triton X-100, splits into two peaks of 9.5 S and 12.5 S in Brij 96-containing gradients. This suggests that hydrophilic G4 BuChE forms are predominant in S1 and that hydrophilic and amphiphilic isoforms coexist in S2.

Altered glycosylation of acetylcholinesterase in lumbar cerebrospinal fluid of patients with Alzheimer's disease

“…The CSF samples were thawed at 4°C, centrifuged (1000× g , 15 min) and aliquots (0.3 ml) added to 0.1 ml (hydrated volume) of Sepharose conjugated lectins (Sigma-Aldrich Pty Ltd, Seven Hills, Australia), either Canavalia ensiformis (Con A) Sepharose or Triticum vulgaris (WGA) Sepharose, incubated at 4°C and unbound AChE assayed at 22°C as previously described10 11 by a modification of the method of Ellman 14…”

“…Gradients contained 50 mM MgCl 2 , 0.5 M NaCl, and 0.5 % (w/v) Triton X-100, which allowed the separation of amphiphilic and non-amphiphilic AChE species as previously described 1112 About 40 fractions were collected and assayed for AChE activity 14. Isoforms of AChE were identified by their sedimentation coefficients using bovine liver catalase (11.4S) and E coli alkaline phosphatase (6.1S) as markers.…”

Acetyl‐ and butyrylcholinesterase activities in the rat retina and retinal pigment epithelium

“…, 1990). A notable proportion of retina AChE (20%) is brought into solution with saline buffer, this percentage of solubilization being similar to that reported in pig (Chai et al, 1981), rat (Vidal et al, 1981), mouse (Vidal et al, manuscript in preparation), and human brain (Saez-Valero et al, 1993). It seems likely that this fraction of soluble enzyme might represent the AChE which is free in the cytosol or looselybound to the cell membranes.…”

Increased Expression of Readthrough Acetylcholinesterase Variants in the Brains of Alzheimer’s Disease Patients

“…Samples (∼0.1 g) of human frontal cortex stored at -80 °C were thawed slowly at 4 °C and homogenized (10% w/v) in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% (w/v) Nonidet P-40, and 0.5% (w/v) Triton X-100 supplemented with a cocktail of protease inhibitors [32]. The homogenates were centrifuged at 70,000 × g at 4 °C for 1 h, and then the supernatants were collected and frozen at -80 °C until assayed.…”

“…AChE activity was determined by a modified microassay version of the colorimetric Ellman's method [32]. AChE was assayed with 1 mM acetylthiocholine and 50 μM tetraisopropyl pyrophosphoramide (Iso OMPA), a specific inhibitor of butyrylcholinesterase, a second cholinesterase that co-exists with AChE in brain.…”