Urs Brodbeck

A rapid and sensitive assay for determination of cholesterol in membrane lipid extracts

A commercially available enzymatic assay (Boehringer Monotest) was modified to allow a rapid and sensitive determination of cholesterol in membrane lipid extracts. This was achieved by adding 0.5% Triton X-100 to the reagent solution. The detergent did not interfere with the assay. The relationship between the amount of cholesterol per assay and the absorbance at 500 nm was linear up to 100 micrograms. The recovery in the assay was better than 95%. The assay was applied to the determination of cholesterol in erythrocyte membrane lipid extracts.

Effect of dimyristoyl phosphatidylcholine on intact erythrocytes. Release of spectrin-free vesicles without ATP depletion

Incubation of human erythrocytes with suspensions of sonicated dimyristoyl phosphatidylcholine resulted in dramatic morphological changes of the cells and release of membrane vesicles. The shedding of membrane vesicles was not preceded by ATP depletion and only occurred at temperatures of incubation that were above the phase transition temperature of dimyristoyl phosphatidylcholine. Membrane vesicles were separated from intact erythrocytes and exogenous dimyristoyl phosphatidylcholine by a series of centrifugation steps. The lipid composition of the membrane vesicles was similar to that of the native erythrocyte, and the predominant membrane proteins were band 3, glycophorin and acetylcholinesterase. Spectrin was not detected. Freeze-fracture electron microscopy showed vesicles (150 nm in diameter) with protein particles embedded in the lipid bilayer.

Tetrameric Detergent-Soluble Acetylcholinesterase from Human Caudate Nucleus: Subunit Composition and Number of Active Sites

Abstract: Purified tetrameric detergent‐soluble acetylcho‐linesterase (DS‐AChE) from human caudate nucleus was analyzed by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis in the absence as well as in presence of a reducing agent. Staining for protein revealed a main band at 66,000 daltons (light monomer) with additional bands at 78,000 daltons (heavy monomer) as well as 130,000 and 150,000 daltons (light and heavy dimers). The same four polypeptides were also detected by Western blotting and by autoradiography of [3H]diisopropylphosphoryl enzyme. Labeling of the enzyme with 3‐trifluoromethyl‐3‐(m‐[125I]‐iodophenyl)diazirine showed that the heavy monomer contained the hydrophobic anchor of the enzyme, whereas the light monomer was practically not labeled. The hydrophobic anchor was susceptible to proteolytic degradation by proteinase K. The functional molarity of DS‐AChE was determined by two independent methods. Four active sites for the tetrameric enzyme were estimated. The turnover number per site was 1.7 ± 107 mol of acetylthiocholine iodide hydrolyzed ± h−1.

Fluorinated aldehydes and ketones acting as quasi-substrate inhibitors of acetylcholinesterase

1. The inhibition of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) by compounds containing trifluoromethyl-carbonyl groups was investigated and related to the effects observed with structurally similar, non-fluorinated chemicals. 2. Compounds that in aqueous solution readily form hydrates inhibit acetylcholinesterase in a time-dependent process. On the other hand non-hydrated, carbonyl-containing compounds showed rapid and reversible, time-independent enzyme inactivation when assayed under steady state conditions. 3. m-N,N,N-Trimethylammonium-acetophenone acts as a rapid and reversible, time-independent, linear competitive inhibitor of acetylcholinesterase (Ki = 5.0 . 10(-7) M). 4. The most potent enzyme inhibitor tested in this series was N,N,N,-trimethylammonium-m-trifluoroacetophenone. It gives time-dependent inhibition and the concentration which inactivates eel acetylcholinesterase to 50% of the original activity after 30 min exposure is 1.3 . 10(-8) M. The bimolecular rate constant for this reaction is 1.8 . 10(6) 1 . mol-1 . min-1. The enzyme-inhibitor complex is very stable as the inhibited enzyme after 8 days of dialysis is reactivated to 20% only. This compound represents a quasi-substrate inhibitor of acetylcholinesterase.

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

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 structural features, suggesting that this novel type of membrane anchor has been conserved during evolution.

Acetylcholinesterase from human erythrocyte membranes: dimers as functional units

Purified human erythrocyte membrane acetylcholinesterase exists as homogenous enzyme species in the presence of detergents. M r estimations of the solubilized enzyme have yielded values from 66 000t>362 000 [1-3]. Most of these analyses have been hampered by the fact that hydrodynamic methods, such as density gradient centrifugation and gel Filtration were used without quantitation of protein-bound detergent. Alternatively, detergent-depleted enzyme forms were used in investigations to elucidate the Mr-values of the resulting multiple aggregates. In these analytical ultracentrifugation studies, 500 0001 137 000 M r has been observed [4]. Yet, no reliable value for the M r of the acetylcholinesterase protomer has been made available. This communication describes the estimation, by a thermodynamic method, of the mass of the enzyme in presence of a detergent which does not affect enzymatic activity and which permits M r determinations without measuring the amount of detergent bound to the protein molecules. The results show that human erythrocyte aeetylcholinesterase in detergent solution exists in a monodisperse state with M r 151 000 + 8000.

Amphiphilic detergent-soluble acetylcholinesterase from Torpedo marmorata: characterization and conversion by proteolysis to a hydrophilic form.

The membrane‐bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was solubilized by Triton X‐100 or by treatment with proteinase K and purified to apparent homogeneity by affinity chromatography. Although the two forms differed only slightly in their subunit molecular weight (66,000 and 65,000 daltons, respectively), considerable differences existed between native and digested detergent‐soluble AChE. The native enzyme sedimented at 6.5 S in the presence of Triton X‐100 and formed aggregates in the absence of detergent. The digested enzyme sedimented at 7.5 S in the absence and in the presence of detergent. In contrast to the detergent‐solubilized AChE, the proteolytically derived form neither bound detergent nor required amphiphilic molecules for the expression of catalytic activity. This led to the conclusion that limited digestion of detergent‐soluble AChE results in the removal of a small hydrophobic peptide which in vivo is responsible for anchoring the protein to the lipid bilayer.

Different glycosylation in acetylcholinesterases from mammalian brain and erythrocytes

Abstract: Acetylcholinesterases (EC 3.1.1.7, AChE) have varying amounts of carbohydrates attached to the core protein. Sequence analysis of the known primary structures gives evidence for several asparagine‐linked carbohydrates. From the differences in molecular mass determined on sodium dodecyl sulfate‐polyacrylamide gel before and after deglycosylation with N‐glycosidase F (EC 3.2.2.18), it is seen that dimeric AChE from red cell membranes is more heavily glycosylated than the tetrameric brain enzyme. Furthermore, dimeric and tetrameric forms of bovine AChE are more heavily glycosylated than the corresponding human enzymes. Monoclonal antibodies 2E6, 1H11, and 2G8 raised against detergent‐soluble AChE from electric organs of Torpedo nacline timilei as well as Elec‐39 raised against AChE from Electrophorus electricus cross‐reacted with AChE from bovine and human brain but not with AChE from erythrocytes. Treatment of the enzyme with N‐glycosidase F abolished binding of monoclonal antibodies, suggesting that the epitope, or part of it, consists of N‐linked carbohydrates. Analysis of N‐acetylglucosamine sugars revealed the presence of N‐acetylglucosamine in all forms of cholinesterases investigated, giving evidence for N‐linked glycosylation. On the other hand, N‐acetylgalactosamine was not found in AChE from human and bovine brain or in butyrylcholinesterase (EC 3.1.1.8) from human serum, indicating that these forms of cholinesterase did not contain O‐linked carbohydrates. Despite the notion that within one species, the different forms of AChE arise from one gene by different splicing, our present results show that dimeric erythrocyte and tetrameric brain AChE must undergo different postsynthetic modifications leading to differences in their glycosylation patterns.

Hydrophobic labeling of the membrane binding domain of acetylcholinesterase from Torpedo marmorata

Membrane‐bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was labeled with the hydrophobic photoactivatable reagent 3‐trifluoromethyl‐3‐(m‐[125I]iodophenyl)diazirine ([125I]TID). Labeling with [125I]TID was restricted to the membranous polypeptide segment of AChE as shown upon conversion of the amphiphilic form to the hydrophilic one by limited digestion with proteinase K. The labeled membranous segment, which has an M r of approx. 3000 was isolated by gel filtration on Sephadex LH‐60 in ethanol/formic acid.

Molecular Forms of Acetylcholinesterase from Human Caudate Nucleus: Comparison of Salt‐Soluble and Detergent‐Soluble Tetrameric Enzyme Species

Abstract: Extraction of human caudate nucleus under high‐ionic‐strength conditions solubilized 20–30% of total acetylcholinesterase (AChE) activity. Density gradient centrifugation revealed monomeric (5.0 S) and tetrameric (11.0 S) enzyme species. The purified, tetrameric salt‐soluble (SS) AChE sedimented at 10.6 S and did not bind detergents. It showed an immunochemical reaction of identity with the detergent‐soluble (DS) AChE species from human caudate nucleus and human erythrocytes, but did not cross‐react with antibodies raised against human serum cholinesterase. The remaining activity was solubilized under low‐ionic‐strength conditions in the presence of 1.0% Triton X‐100. The purified tetrameric, DS‐AChE sedimented at 10.0 S as detergent‐protein mixed micelle and on extensive removal of the detergent this enzyme formed defined aggregates by self‐micellarization. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under nonreducing conditions revealed that the salt‐soluble and detergent‐soluble tetrameric enzyme species both contained a heavy and a light dimer; under reducing conditions mainly one band corresponding to the light subunit was seen. Molecular weights of 300,000 dalton and 280,000 dalton were calculated for SS‐AChE and DS‐AChE, respectively. Limited digestion of DS‐AChE with proteinase K led to isolation of an enzyme that no longer bound detergents and lacked the inter‐subunit disulfide bridges.