The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

1987 ◽  
Vol 65 (1) ◽  
pp. 8-18 ◽  
Author(s):  
Rex K. M. Wong ◽  
Christine P. Nichol ◽  
M. Chandra Sekar ◽  
Basil D. Roufogalis
Keyword(s):  
Chain Length ◽  
Membrane Lipids ◽  
Acetylcholinesterase Activity ◽  
Exchange Chromatography ◽  
Erythrocyte Membranes ◽  
Tween 20 ◽  
Bovine Erythrocyte ◽  
Critical Micelle Concentrations ◽  
Triton X ◽  
Nonionic Detergents

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octy1 β-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70–80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

1991 ◽  
Vol 69 (2-3) ◽  
pp. 154-162 ◽  
Author(s):  
Christine P. Nichol ◽  
Basil D. Roufogalis
Keyword(s):  
Phase Separation ◽  
Affinity Chromatography ◽  
Phospholipase C ◽  
Lectin Binding ◽  
Acetylcholinesterase Activity ◽  
Erythrocyte Membranes ◽  
Bovine Erythrocyte ◽  
Hydrophobic Properties ◽  
Native Form ◽  
Triton X

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by α-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.Key words: acetylcholinesterase, bovine erythrocytes, phosphatidylinositol-specific phospholipase C, phase separation, affinity chromatography.

The inhibitory effects of polyoxyethylene detergents on human erythrocyte acetylcholinesterase and Ca2+ + Mg2+ ATPase

1989 ◽  
Vol 67 (2-3) ◽  
pp. 137-146 ◽  
Author(s):  
R. Blaine Moore ◽  
J. F. Manery ◽  
J. Still ◽  
V. N. Mankad
Keyword(s):  
Human Erythrocyte ◽  
Acetylcholinesterase Activity ◽  
Significant Inhibition ◽  
Erythrocyte Membranes ◽  
Head Group ◽  
Polar Head Group ◽  
Detergent Concentration ◽  
Human Erythrocyte Membranes ◽  
Hydrophobic Tail ◽  
Triton X

The activities of acetylcholinesterase and Ca2+ + Mg2+ ATPase were measured following treatment of human erythrocyte membranes with nonsolubilizing and solubilizing concentrations of Triton X-100. A concentration of 0.1% (v/v) Triton X-100 caused a significant inhibition of both enzymes. The inhibition appears to be caused by perturbations in the membrane induced by Triton X-100 incorporation. No acetylcholinesterase activity and little Ca2+ + Mg2+ ATPase activity were detected in the supernatant at 0.05% Triton X-100 although this same detergent concentration induced changes in the turbidity of the membrane suspension. Also, no inhibition of soluble acetylcholinesterase was observed over the entire detergent concentration range. The inhibition of these enzymes at 0.1% Triton X-100 was present over an eightfold range of membrane protein in the assay indicating an independence of the protein/detergent ratio. The losses in activities of these two enzymes could be prevented by either including phosphatidylserine in the Triton X-100 suspension or using Brij 96 which has the same polyoxyethylene polar head group but an oleyl hydrophobic tail instead of the p-tert-octylphenol group of Triton X-100. The results are discussed in regard to the differential recovery of enzyme activities over the entire detergent concentration range.Key words: Triton X-100, erythrocyte membranes, acetylcholinesterase, Ca2+ + Mg2+ ATPase, polyoxyethylene detergents.

Detergent Modification of Membranes: Fact or Artifact?

1985 ◽  
Vol 43 ◽  
pp. 558-559
Author(s):  
Margaret E. Hogan ◽  
Karen K. Baker ◽  
Stanley Flegler
Keyword(s):  
Electron Microscopy ◽  
Critical Micelle Concentration ◽  
Lipid Bilayer ◽  
Rat Liver ◽  
Tween 20 ◽  
Isolated Rat Liver ◽  
Critical Micelle Concentrations ◽  
Liver Nuclei ◽  
Triton X ◽  
Isolated Rat

Detergents have been used to modify biological membranes from nuclear envelopes and organelles to cellular bilayers of many cultured organisms. These modifications, usually designed to permeabilize or strip away the lipid bilayer, are as varied as the types of detergents used. The most commonly used agents are those with low critical micelle concentrations (Triton X-100, Tween-20, NP-40) making them difficult to completely remove from the reaction mixture. Octyl-POE (polydisperse octyloligooxyethylene), a detergent of high critical micelle concentration, is dialyzable and has been used in hopes of eliminating the possible masking effects of residual detergent in electron microscopy preparations. Through the use of a common treatment for all test solutions, a comparison of Triton X-100, NP-40, octyl-POE and a buffered solution as a control has been performed on isolated rat liver nuclei.

Antioxidative Properties Of Pyrrolidinium And Piperidinium Salts

2002 ◽  
Vol 57 (3-4) ◽  
pp. 344-347 ◽  
Author(s):  
Halina Kleszczyńska ◽  
Małgorzata Oświęcimska ◽  
Dorota Bonarska ◽  
Janusz Sarapuk
Keyword(s):  
Chain Length ◽  
Membrane Lipids ◽  
Hydrocarbon Chain ◽  
Erythrocyte Membranes ◽  
Lipid Phase ◽  
Rbc Membrane ◽  
Hydrocarbon Chain Length ◽  
Antioxidant Function ◽  
Different Depths ◽  
The Difference

Two series of pyrrolidinium (PYA-n) and piperidinium (PPPA-n) bromides with incorporated antioxidant function were synthesized. Both have hydrocarbon chains with odd number of the carbon atoms (n) ranging between 7 and 15. Pig erythrocytes (RBC) were used to study antioxidant activity of these compounds. They were incorporated into RBC membranes in sublytic (micromolar) concentrations and RBC were then subjected to UV radiation. It was found that all the salts used protected erythrocyte membranes against oxidation of membrane lipids. This protection increased with hydrocarbon chain length. Such effect may be the result of an incorporation of particular compounds to different depths into the lipid phase of RBC membrane depending on their chain length. Such possibility was checked by studies on fluidity changes induced by the compounds studied in ghost membranes by fluorimetric measurements. The measurements showed that pyrrolidinium bromides were slightly more effective in a protection of erythrocytes than the corresponding piperidinium ones. The possible reason of such behaviour may be the difference in lipophilicity between piperidine and pyrrolidine rings

Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria

1977 ◽  
Vol 55 (10) ◽  
pp. 1114-1117 ◽  
Author(s):  
Gerrit H. Bomhoff ◽  
Mary Spencer
Keyword(s):  
Ionic Strength ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Ferrocytochrome C ◽  
Optimum Ph ◽  
Triton X ◽  
Assay Conditions ◽  
Nonionic Detergents

Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents Triton X-114 and Triton X-100, from pea cotyledon mitochondria. Optimum assay conditions were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.

scholarly journals Ultrastructure of the intact skeleton of the human erythrocyte membrane.

1986 ◽  
Vol 102 (3) ◽  
pp. 997-1006 ◽  
Author(s):  
B W Shen ◽  
R Josephs ◽  
T L Steck
Keyword(s):  
Human Erythrocyte ◽  
Actin Filaments ◽  
Band 3 ◽  
Carbon Films ◽  
Erythrocyte Membranes ◽  
Accessory Proteins ◽  
Red Cell Membrane ◽  
Human Erythrocyte Membranes ◽  
Low Ionic Strength ◽  
Triton X

Filamentous skeletons were liberated from isolated human erythrocyte membranes in Triton X-100, spread on fenestrated carbon films, negatively stained, and viewed intact and unfixed in the transmission electron microscope. Two forms of the skeleton were examined: (a) basic skeletons, stripped of accessory proteins with 1.5 M NaCl so that they contain predominantly polypeptide bands 1, 2, 4.1, and 5; and (b) unstripped skeletons, which also bore accessory proteins such as ankyrin and band 3 and small plaques of residual lipid. Freshly prepared skeletons were highly condensed. Incubation at low ionic strength and in the presence of dithiothreitol for an hour or more caused an expansion of the skeletons, which greatly increased the visibility of their elements. The expansion may reflect the opening of spectrin from a compact to an elongated disposition. Expanded skeletons appeared to be organized as networks of short actin filaments joined by multiple (5-8) spectrin tetramers. In unstripped preparations, globular masses were observed near the centers of the spectrin filaments, probably corresponding to complexes of ankyrin with band 3 oligomers. Some of these globules linked pairs of spectrin filaments. Skeletons prepared with a minimum of perturbation had thickened actin protofilaments, presumably reflecting the presence of accessory proteins. The length of these actin filaments was highly uniform, averaging 33 +/- 5 nm. This is the length of nonmuscle tropomyosin. Since there is almost enough tropomyosin present to saturate the F-actin, our data support the hypothesis that tropomyosin may determine the length of actin protofilaments in the red cell membrane.

scholarly journals Mucin biosynthesis. Properties of a bovine tracheal mucin β-6-N-acetylglucosaminyltransferase

1985 ◽  
Vol 227 (2) ◽  
pp. 405-412 ◽  
Author(s):  
P W Cheng ◽  
W E Wingert ◽  
M R Little ◽  
R Wei
Keyword(s):  
Enzyme Activity ◽  
Freezing Point ◽  
Cetylpyridinium Chloride ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Gas Chromatography Mass Spectrometry ◽  
Tween 20 ◽  
Group A ◽  
Michaelis Constants ◽  
Triton X

We have characterized a bovine tracheal mucin beta-6-N-acetylglucosaminyltransferase that catalyses the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to the C-6 of the N-acetylgalactosamine residue of galactosyl-β 1→3-N-acetylgalactosamine. Optimal enzyme activity was obtained between pH 7.5-8.5, at 5mM-MnCl2, and at 0.06-0.08% (v/v) Triton X-100 (or Nonidet P-40), or 0.5-5.0% (v/v) Tween 20. Ba2+, Mg2+ and Ca2+ could partially replace Mn2+, but Co2+, Fe2+, Cd2+ and Zn2+ could not. Sodium dodecyl sulphate, cetylpyridinium chloride, sodium deoxycholate, octyl beta-D-glucoside, digitonin and alkyl alcohols were less effective in enhancing enzyme activity, and dimethyl sulphoxide was ineffective. The apparent Michaelis constants were 1.25 mM for UDP-N-acetylglucosamine, 0.94-3.34 mM for freezing-point-depressing glycoprotein and 0.19 mM for periodate-treated blood-group-A porcine submaxillary mucin. Asialo ovine submaxillary mucin could not serve as the glycosyl acceptor. The structure of the 14C-labelled oligosaccharide obtained by alkaline-borohydride treatment of the product was identified as Gal beta 1→3(Glc-NAc beta 1→6)N-acetylgalactosaminitol by beta-hexosaminidase treatment, gas chromatography-mass spectrometry and 1H-n.m.r. (270 MHz) analysis. The enzyme is important in the regulation of mucin oligosaccharide biosynthesis.

Transmembrane phospholipid distribution revealed by freeze-fracture replica labeling

1996 ◽  
Vol 109 (10) ◽  
pp. 2453-2460 ◽  
Author(s):  
K. Fujimoto ◽  
M. Umeda ◽  
T. Fujimoto
Keyword(s):  
Plasma Membranes ◽  
Membrane Lipids ◽  
Secretory Granules ◽  
General Scheme ◽  
Sodium Dodecyl ◽  
Freeze Fracture ◽  
Electron Microscopic Observation ◽  
Erythrocyte Membranes ◽  
Electron Microscopic ◽  
Intracellular Membranes

We propose the use of membrane splitting by freeze-fracture for differential phospholipid analysis of protoplasmic and exoplasmic membrane leaflets (halves). Unfixed cells or tissues are quick-frozen, freeze-fractured, and platinum-carbon (Pt/C) shadowed. The Pt/C replicas are then treated with 2.5% sodium dodecyl sulfate (SDS) to solubilize unfractured membranes and to release cytoplasm or contents. While the detergent dissolves unfractured membranes, it would not extract lipids from split membranes, as their apolar domains are stabilized by their Pt/C replicas. After washing, the Pt/C replicas, along with attached protoplasmic and exoplasmic membrane halves, are processed for immunocytochemical labeling of phospholipids with antibody, followed by electron microscopic observation. Here, we present the application of the SDS-digested freeze-fracture replica labeling (SDS-FRL) technique to the transmembrane distribution of a major membrane phospholipid, phosphatidylcholine (PC), in various cell and intracellular membranes. Immunogold labeling revealed that PC is exclusively localized on the exoplasmic membrane halves of the plasma membranes, and the intracellular membranes of various organelles, e.g. nuclei, mitochondria, endoplasmic reticulum, secretory granules, and disc membranes of photoreceptor cells. One exception to this general scheme was the plasma membrane forming the myelin sheath of neurons and the Ca(2+)-treated erythrocyte membranes. In these cell membranes, roughly equal amounts of immunogold particles for PC were seen on each outer and inner membrane half, implying a symmetrical transmembrane distribution of PC. Initial screening suggests that the SDS-FRL technique allows in situ analysis of the transmembrane distribution of membrane lipids, and at the same time opens up the possibility of labeling membranes such as intracellular membranes not normally accessible to cytochemical labels without the distortion potentially associated with membrane isolation procedures.

scholarly journals Glycoprotein V is not the thrombin activation receptor on human blood platelets

Blood ◽  
1986 ◽  
Vol 68 (3) ◽  
pp. 720-725 ◽  
Author(s):  
D Bienz ◽  
W Schnippering ◽  
KJ Clemetson
Keyword(s):  
Platelet Activation ◽  
Polyclonal Antibodies ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Exchange Chromatography ◽  
Thrombin Activation ◽  
Strong Candidate ◽  
Triton X ◽  
Kinetics Of

Thrombin activation of platelets involves two receptors: glycoprotein Ib (GPIb), which affects the kinetics of the response; and, as a strong candidate for the second, essential receptor, GPV, a hydrophobic, 82-kd glycoprotein with an isoelectric point (pI) of pH 5.85 to 6.55. Whole platelets were treated with endogenous platelets calcium-activated proteases, yielding a major fragment, GPV8, with molecular weight (mol wt) of 79 kilodaltons (kd). The fragment was purified by affinity chromatography on wheat germ agglutinin followed by ion exchange chromatography on DEAE-Sephacel using first a 0 to 0.7-mol/L and then a 0 to 0.3-mol/L NaCl gradient. A rabbit was immunized with the purified GPV8 for preparation of polyclonal antibodies. Crossed immunoelectrophoresis and two-dimensional polyacrylamide gel electrophoresis (PAGE) electrophoretic blotting with the separate phases of a Triton X-114 phase partition of human platelets showed the characteristic pattern of GPV in the hydrophobic phase. During thrombin- induced platelet aggregation GPV is hydrolysed, releasing a fragment, GPVf1, to the supernatant. The fragment GPVf1 still contains a thrombin- binding site. Anti-GPV antibodies blocked GPV proteolysis, but did not inhibit platelet activation induced by thrombin. We conclude that proteolysis of GPV by thrombin is not essential for platelet activation.

Protective Effect of Quaternary Piperidinium Salts on Lipid Oxidation in the Erythrocyte Membrane

1999 ◽  
Vol 54 (5-6) ◽  
pp. 424-428 ◽  
Author(s):  
Halina Kleszczyńska ◽  
Małgorzata Oświęcimska ◽  
Janusz Sarapuk ◽  
Stanisław Przestalski ◽  
Stanisław Witek
Keyword(s):  
Lipid Oxidation ◽  
Chain Length ◽  
Erythrocyte Membrane ◽  
Alkyl Chain ◽  
Alkyl Chain Length ◽  
Erythrocyte Membranes ◽  
Negative Consequences ◽  
Rosa Rugosa ◽  
Antioxidant Efficiency ◽  
Comparison Of The Results

Abstract A new series of amphiphilic compounds with incorporated antioxidant functional group has been investigated. Piperidinium bromides, differing in the alkyl chain length (8, 10, 12, 14 and 16 carbon atoms in the chain) were synthesised to protect biological and/or model membranes against peroxidation and following negative consequences. Their antioxidant activity was studied with erythrocytes subjected to UV radiation. The salts used inhibited lipid oxidation in the erythrocyte membrane. The degree of this inhibition depended on the alkyl chain length of the bromide used and increased with increasing alkyl chain length. A comparison of the results obtained for piperidinium bromides with those obtained for the widely used antioxidant 3,5-di-t-butyl-4-hydroxytoluene (BHT) revealed that only two shortest alkyl chain salts were less efficient than BHT in protecting erythrocyte membranes. A similar comparison with antioxidant efficiency of flavonoids extracted from Rosa rugosa showed that they protected the membranes studied more weakly than the least effective eight-carbon alkyl chain piperidinium bromide. The three compounds of longest alkyl chains were the most active antioxidants. Their activities did not differ significantly.

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