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 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.

Resistance of Human Erythrocyte Membranes to Triton X-100 and C12E8

2008 ◽  
Vol 227 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Cleyton Crepaldi Domingues ◽  
Annarita Ciana ◽  
Armando Buttafava ◽  
Cesare Balduini ◽  
Eneida de Paula ◽  
...  
Keyword(s):  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membranes ◽  
Triton X

scholarly journals Properties of particulate, membrane-associated and soluble guanylate cyclase from cardiac muscle, skeletal muscle, cerebral cortex and liver

1976 ◽  
Vol 157 (3) ◽  
pp. 713-719 ◽  
Author(s):  
S J Sulakhe ◽  
N L Leung ◽  
P V Sulakhe
Keyword(s):  
Guanylate Cyclase ◽  
Metal Binding ◽  
Plasma Membranes ◽  
Soluble Guanylate Cyclase ◽  
Inhibitory Effect ◽  
Ion Concentration ◽  
Metal Binding Sites ◽  
Microsomal Membranes ◽  
Solubilized Enzyme ◽  
Triton X

1. Guanylate cyclase of washed particles and plasma membranes showed S-shaped progress curves when titrated with either GTP or Mn2+ ions; similar results were obtained with Triton X-100-solubilized enzyme preparation from washed particles. Hill plots of these data revealed multiple metal-nucleotide and free-metal binding sites. 2. Guanylate cyclase of supernatant fractions displayed typical Michaelis-Menten properties when enzyme required excess of (free) Mn2+ (over GTP) for maximal activities; Ka (free Mn2+) was about 0.15-0.25 mM at subsaturating concentrations of GTP. 4 MnATP, MnADP, and MnGDP were found to increase the activities of both particulate and superantant enzyme, when MnGTP concentration was below saturation and free Mn2+ ion concentration was low (less than 100 muM); MnATP (50muM-1 mM) inhibited both these activities at high free Mn2+ concentration (1.5 mM) and inhibition of the particulate enzyme was greater than that of supernatant enzyme. 5. Ca2+ ions stimulated supernatant-enzyme activity; the stimulatory concentration of Ca2+ ions depended on the concentration of Mn2+ and GTP. 6. A modest stimulation of particulate guanylate cyclase by pyrophosphate (0.02-1 mM) was observed; the pyrophosphate effect appeared to be competitive with respect to GTP. At a higher concentration (2 mM), pyrophosphate produced a marked inhibition of particulate enzyme; the nature of inhibitory effect appeared complex. 7. Inorganic salts (e.g. NaCl, KCl, LiBr, NaF) produced inhibition of particulate enzyme; the degree of inhibition of Triton X-100-stimulated activity was less than that of unstimulated activity. 9. Treatment of sarcolemmal or microsomal membranes with either phospholipase C or trypsin decreased, whereas phospholipase A increased, the activity of guanylate cyclase.

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.

Filaments Associated With the Human Erythrocyte Membrane: A Scanning Electron Microscope Study

1976 ◽  
Vol 34 ◽  
pp. 28-29
Author(s):  
J. F. Hainfeld
Keyword(s):  
Electron Microscope ◽  
Scanning Electron Microscope ◽  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Interior Space ◽  
Intact Cells ◽  
Scanning Electron Microscope Study ◽  
Human Erythrocyte Membranes ◽  
Triton X ◽  
Scanning Electron

A reticulum of filaments covering the cytoplasmic surface of human erythrocyte membranes was visualized at a resolution of 50-100 Å using a scanning electron microscope. This network was visible in ghosts split or torn open to reveal their interior space; in Triton X-100 extracted ghost residues; and even in intact cells, where the contour of the outer surfaces appeared to reflect an underlying meshwork. In addition to filaments, annular figures and nodes were seen in the reticulum. Since the Triton X-100 extraction leaves insoluble a residue that is predominantly spectrin and actin, the residues observed may be assumed to be composed of these proteins. Also, since the reticulum seen inside ghosts appears morphologically similar to the Triton residue reticulum, it may be tentatively concluded that this, too, is made up of spectrin and actin.

scholarly journals Are polyphosphoinositides associated with glycophorin in human erythrocyte membranes?

1979 ◽  
Vol 179 (2) ◽  
pp. 441-444 ◽  
Author(s):  
S D Shukla ◽  
R Coleman ◽  
J B Finean ◽  
R H Michell
Keyword(s):  
Erythrocyte Membrane ◽  
Human Erythrocyte ◽  
Erythrocyte Membranes ◽  
Human Erythrocyte Membrane ◽  
Human Erythrocyte Membranes ◽  
Partition Method ◽  
Triton X

Glycophorin prepared by a lithium di-iodosalicylate-extraction/phenol-partition method was rich in polyphosphoinositides (phosphatidyl-myo-inositol 4-phosphate and phosphatidyl-myo-inositol 4,5-bisphosphate), but glycophorin extracted by Triton X-100 showed no such enrichment. The enrichment observed in the former preparations appeared not to be caused by pre-existing association between glycophorin and polyphosphoinositides in the human erythrocyte membrane, but to be largely a consequence of the preparative procedures.

Phospholipid and calmodulin activation of solubilized calcium-transport ATPase from human erythrocytes: regulation by magnesium

1981 ◽  
Vol 59 (11-12) ◽  
pp. 880-888 ◽  
Author(s):  
Ameera Al-Jobore ◽  
Basil D. Roufogalis
Keyword(s):  
Fatty Acids ◽  
Atpase Activity ◽  
Calcium Transport ◽  
Erythrocyte Membranes ◽  
Transport Atpase ◽  
Partial Reaction ◽  
Low Concentrations ◽  
Human Erythrocyte Membranes ◽  
Solubilized Enzyme ◽  
Triton X

The effect of phospholipids on Triton X-100 solubilized (Ca2+ + Mg2+)-ATPase from human erythrocyte membranes has been examined. The enzyme activity was increased by phosphatidylinositol, phosphatidylserine, and phosphatidic acid at both low (2 μM) and high (65 μM) free Ca2+ concentrations, while phosphatidylcholine had little effect and phosphatidyiethanolamine and cardiolipin inhibited the (Ca2+ + Mg2+)-ATPase activity at all Ca2+ concentrations studied. The diacylglycerol, diolein, inhibited the enzyme at high, but not low, Ca2+ concentrations. Low concentrations of phospholipase A2 (1–2 international units) also activated the solubilized enzyme, at least in part by releasing free fatty acids, as the activation was mimicked by oleic acid (1–2 μmol/mg protein) and was abolished by fatty acid depleted bovine serum albumin. The combined activation by saturating levels of phosphatidylserine and calmodulin was additive at 6.5 mM MgCl2, and probably occurred at distinct sites on a regulatory component of the enzyme. The activation by both effectors was antagonized by MgCl2 from 0.5 to 6.5 mM. Ca2+ inhibition of the enzyme was also antagonized by MgCl2 at similar concentrations. Analysis of various models suggested that phosphatidylserine had two effects on (Ca2+ + Mg2+)-ATPase activity. First, a low Ca2+ affinity form of the enzyme was converted to a high Ca2+ affinity form, which was more sensitive to Ca2+ inhibition. Second, it increased the turnover of the enzyme, probably by enhancing its dephosphorylation, which was mimicked in this study by the Ca2+-dependent p-nitrophenyiphosphatase partial reaction.

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