scholarly journals A study of the mechanism by which some amphiphilic drugs affect human erythrocyte acetylcholinesterase activity

1989 ◽  
Vol 261 (2) ◽  
pp. 569-573 ◽  
Author(s):  
A Spinedi ◽  
L Pacini ◽  
P Luly
Keyword(s):  
Human Erythrocyte ◽  
Physical State ◽  
Membrane Integrity ◽  
Acetylcholinesterase Activity ◽  
Inhibition Kinetics ◽  
Inhibitory Potency ◽  
Mixed Inhibition ◽  
Amphiphilic Drugs ◽  
Lipid Environment ◽  
Triton X

The effects of the local anaesthetics procaine, tetracaine and lidocaine and of the antidepressant imipramine on human erythrocyte acetylcholinesterase were investigated. All four amphiphilic drugs inhibited enzymic activity, the IC50 (the concentration causing 50% inhibition) being about 0.40 mM for procaine, 0.05 mM for tetracaine, 0.70 mM for imipramine and 7.0 mM for lidocaine. Procaine and tetracaine inhibited acetylcholinesterase activity competitively at concentrations at which they did not perturb the physical state of the membrane lipid environment, as assessed by steady-state fluorescence polarization, whereas lidocaine and imipramine displayed mixed inhibition kinetics at concentrations at which they induced an enhancement of membrane fluidity. The question was addressed as to whether membrane integrity is a prerequisite for imipramine and lidocaine action. Membrane solubilization by 1% Triton X-100 and a decrease, by dilution, in the detergent concentration to 0.05% [which is above the Triton X-100 critical micelle concentration (c.m.c.)] did not substantially affect the inhibitory potency of the two amphiphilic drugs at their IC50; in the presence of increasing detergent concentrations the inhibitory potency of imipramine was gradually decreased, but not abolished, whereas the inhibitory effect of lidocaine was only slightly diminished, even at 1% Triton X-100. It is suggested that neither competitive nor mixed inhibition kinetics arise from conformational changes of the protein driven by a modification of the physical state of the lipid environment, but from a direct interaction of the amphiphilic drugs with acetylcholinesterase. In particular, the partial loss of the inhibitory potency of imipramine and lidocaine that is observed upon increasing Triton X-100 concentration well above its c.m.c. could be explained in terms of amphiphile partition in detergent micelles and, in turn, of a decreased effective concentration of the two inhibitors in the aqueous phase.

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.

scholarly journals A study of human erythrocyte acetylcholinesterase inhibition by chlorpromazine

1991 ◽  
Vol 278 (2) ◽  
pp. 461-463 ◽  
Author(s):  
A Spinedi ◽  
L Pacini ◽  
C Limatola ◽  
P Luly ◽  
R N Farias
Keyword(s):  
Human Erythrocyte ◽  
Inhibitory Effect ◽  
Membrane Lipid ◽  
Acetylcholinesterase Inhibition ◽  
Erythrocyte Membranes ◽  
Micellar Aggregates ◽  
Solubilized Enzyme ◽  
Erythrocyte Ache ◽  
Lipid Environment ◽  
Triton X

Membrane-bound acetylcholinesterase (AChE) from the human erythrocyte is inhibited by chlorpromazine (CPZ) in a concentration range within this amphiphilic drug has been demonstrated to interact with erythrocyte membranes, causing a large spectrum of physical and structural effects; membrane solubilization with 0.5% Triton X-100 results in a complete loss of CPZ inhibitory potency. Although these observations might suggest a role of membrane lipid environment in mediating human erythrocyte AChE inhibition, we observed that CPZ retains its full inhibitory effect on the fraction of enzyme (5-6% of total) that is solubilized from erythrocytes upon treatment with phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis; furthermore, Triton X-100 is able to reverse the CPZ effect also in the case of PI-PLC-solubilized enzyme. These results demonstrate unequivocally that CPZ inhibits human erythrocyte AChE through direct molecular interaction. The inhibition kinetics displayed by CPZ on human erythrocyte AChE are dependent on drug concentration: evidence is provided that this phenomenon may be related to formation of CPZ micellar aggregates.

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.

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.

scholarly journals Protein chromatography on adsorbents with hydrophobic and ionic groups. Purification of human erythrocyte glycophorin

1977 ◽  
Vol 163 (2) ◽  
pp. 397-400 ◽  
Author(s):  
R J Simmonds ◽  
R J Yon
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Human Erythrocyte ◽  
Sodium Dodecyl ◽  
Erythrocyte Ghosts ◽  
Electrophoretic Band ◽  
Protein Chromatography ◽  
Ionic Groups ◽  
A Subunit ◽  
Triton X

Human erythrocyte glycophorin was purified rapidly by (a) chromatography of a Triton X-100 extract of erythrocyte ‘ghosts’ on N-(3-carboxypropionyl)aminodecyl-Sepharose in buffers containing Triton X-100 or sodium dodecyl sulphate, or (b) chromatography of whole ‘ghosts’, solubilized in sodium dodecyl sulphate, on dodecyl-Sepharose, in buffers containing sodium dodecyl sulphate. The products contained 85-95% glycophorin (electrophoretic band PAS-1) and the major contaminants were glycoproteins PAS-2 (possibly a subunit of glycophorin) and PAS-3.

scholarly journals Solubilization of human erythrocyte membrane glycoproteins by triton X-100

1979 ◽  
Vol 179 (2) ◽  
pp. 299-303 ◽  
Author(s):  
R S Pratt ◽  
G M Cook
Keyword(s):  
Sialic Acid ◽  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
General Procedure ◽  
Membrane Glycoprotein ◽  
Membrane Glycoproteins ◽  
Human Erythrocyte Membrane ◽  
Ionic Detergent ◽  
Triton X ◽  
Mild Solubilization

1. The enzymic removal of sialic acid residues from the glycoproteins of the human erythrocyte decreases the solubilization of membrane glycoprotein by Triton X-100. 2. The solubilization of asialoglycoprotein by Triton X-100 may be restored by the addition of borate. 3. Use of this non-ionic detergent in the presence of borate, as a general procedure for the mild solubilization of membrane glycoproteins deficient in sialic acid residues, is discussed.

scholarly journals Phosphotyrosine phosphatase associated with band 3 protein in the human erythrocyte membrane

1996 ◽  
Vol 314 (3) ◽  
pp. 881-887 ◽  
Author(s):  
Yehudit ZIPSER ◽  
Nechama S. KOSOWER
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Intact Cell ◽  
Band 3 ◽  
Band 3 Protein ◽  
Human Erythrocyte Membrane ◽  
Major Fraction ◽  
Phosphotyrosine Phosphatase ◽  
A Minor ◽  
Triton X

The anion-exchange band 3 protein is the main erythrocyte protein that is phosphorylated by tyrosine kinase. To study the regulation of band 3 phosphorylation, we examined phosphotyrosine phosphatase (PTP) activity in the human erythrocyte. We show that the human erythrocyte membrane contains a band 3-associated neutral PTP which is activated by Mg2+ and inhibited by Mn2+ and vanadate. The PTP is active in the intact cell and in the isolated membrane. A major fraction of the PTP is tightly bound to the membrane and can be extracted from it by Triton X-100; a minor part is associated with the Triton X-100-insoluble cytoskeleton. The behaviour of the PTP parallels that of band 3, the major fraction of which is extractable by detergents with a minor fraction being anchored to the cytoskeleton. Moreover, band 3 is co-precipitated when the PTP is immunoprecipitated from solubilized membranes, and PTP is co-precipitated when band 3 is immunoprecipitated. The PTP appears to be related to PTP1B (identified using an antibody to an epitope in its catalytic domain and by molecular mass). The system described here has a unique advantage for PTP research, since it allows the study of the interaction of a PTP with an endogenous physiological substrate that is present in substantial amounts in the cell membrane. The membrane-bound, band 3-associated, PTP may play a role in band 3 function in the erythrocyte and in other cells which have proteins analogous to band 3.

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