Amphiphile-induced vesiculation in aged hereditary spherocytosis erythrocytes indicates normal membrane stability properties under non-starving conditions

Aged HS erythrocytes with a defined primary defect in band 3 protein or ankyrin were incubated with amphiphiles (detergents) at sublytic concentrations (37° C, 60 min) or glucose-starved (37° C, 24 h). In line with previous studies, the release of AChE (exovesicles) from HS erythrocytes during glucose-starvation was significantly higher (11%) compared to that from control erythrocytes (1%). Control and HS cells responded, however, similarly to amphiphile-treatment (non-starving conditions). Amphiphiles induced similar types of shape alterations and a similar amount of AChE release (14- 15%). Furthermore, the size and shape of amphiphile-induced exo- and endovesicles released from control and HS erythrocytes were similar. The results suggest that the stability properties of the membrane are not seriously disturbed in aged HS erythrocytes under nonstarving conditions.     

GPI-linked proteins do not transfer spontaneously from erythrocytes to liposomes. New aspects of reorganization of the cell membrane

Exposure of cells to liposomes results in the release of integral membrane proteins. However, it is still controversial whether the release is due to spontaneous protein transfer from cells to liposomes or shed vesicles released from cells. We investigated this issue in an erythrocyte-liposome system by examining the location of acetylcholinesterase (AChE, an integral membrane protein marker), cholesterol (erythrocyte membrane lipid marker), hemoglobin (cytosolic protein marker), and a nonexchangeable lipid marker in liposomes in a sucrose density gradient at high resolution. The density distribution showed that AChE is not transferred to the liposomes but is located on small (about 50 nm) light (10-20 wt % sucrose) or large (about 200 nm) heavy shed vesicles (more than 30 wt % sucrose). AChE in the light shed-vesicle fraction markedly increased even after its level in the heavy fraction reached a plateau. AChE was also released from isolated heavy shed vesicles and accumulated in the small light shed-vesicle fraction in the presence of liposomes. After incubation of spherical erythrocytes (morphological index, 5.0) with liposomes, AChE hardly appeared in the heavy shed-vesicle fraction, and the majority (>99%) appeared in the light shed-vesicle fraction, indicating that AChE is released from both the erythrocytes and heavy shed vesicles to the light shed-vesicle fraction, which becomes rich in AChE. Our results demonstrated for the first time that GPI-linked proteins do not spontaneously transfer from erythrocytes to liposomes. Our study also suggests that in vivo GPI-linked membrane proteins do not spontaneously transfer between cell membranes but that some catalyst is needed.     

Induction of acetylcholinesterase release from erythrocytes in the presence of liposomes

When human erythrocytes are incubated with liposomes, the release of acetylcholinesterase (AChE) occurs following an induction period [Cook et al. (1980) Biochemistry 19, 4601-4607]. However, the mechanism of the induction has not been elucidated. We examined the relationships among the lipid transfer from liposomes to erythrocytes, the morphological change of erythrocytes, the fluidity of the erythrocyte membrane and the start of AChE release. The AChE release into the liposomes and into shed-vesicle fractions started simultaneously after an induction period. The morphological index (MI) of erythrocytes was approximately 2.8 at the beginning of the release, regardless of the induction period. AChE was not released from the erythrocytes of index 2.8 even in the presence of liposomes if the MI remained at 2.8. Therefore, for the release, erythrocytes needed a further increase of the MI from 2.8. As the rate of lipid transfer increased, the induction period became shorter. No significant lipid release from erythrocytes was detected during the induction period. The initiation of the AChE release was not simply affected by the change in the membrane fluidity of erythrocytes upon interaction with liposomes. These results first demonstrate that AChE release into the shed-vesicle and liposome fractions is triggered by a further increase of the MI from 2.8, which is induced by lipid transfer from liposomes to erythrocytes.     

Molecular forms of acetylcholinesterase: their de novo synthesis in mouse neuroblastoma cells

Rat mouse AChE molecular forms are indistinguishable with respect to their sedimentation coefficients and their evolutive proportions during brain maturation. Among rat or mouse erythrocytes, rat C6 glial cells, and mouse 2A and NS 20 neuroblastoma cells, only neuroblastoma cells showed both the ES and HS molecular forms with a 1:1 proportion for NS 20 cells. All these cells lack a third molecular form (16S), which is present in rat and mouse superior cervical ganglia. After irreversible inhibition of pre-existing NS 20 neuroblastoma AchE, the ES form is first synthesized (de novo synthesis). The HS form begins to appear after a lag time of several hours and represents, 24 h after inhibition, only 15% of the total recovered activity, which is near the initial level. The initial relative proportions return by 2 to 3 days after inhibition. The recovery of the HS form is, for the most part, blocked by actinomycin D, which does not block the recovery of activity itself, which remains as an ES form. It seems that integration of the ES form into the HS form more probably depends on the synthesis of a new messenger RNA, which is required for the synthesis of either new AChE polypeptide chain, polymerization initiating protein or activating enzyme.     

Calcium release of heat-shocked porcine oocytes induced by thimerosal or inositol 1,4,5-trisphosphate (IP3)

The objective of this study was to determine the effect of heat shock (HS) on the Ca(2+) release and the subsequent development in matured porcine oocytes. Oocytes were matured in vitro and randomly allocated to different heat treatments at 41.5 degrees C for 1 (HS1h), 2 (HS2h) or 4h (HS4h). Control groups of oocytes were cultured for 0 or 4h without HS (39 degrees C, C0h, C4h). In Experiment 1 (eight replicates), matured oocytes were activated by thimerosal (200 microM, 10 min) following HS. Among all heated groups, maximal intracellular calcium concentration ([Ca(2+)](i)) was the highest in the HS2h. The lowest [Ca(2+)](i) peak among HS groups was observed in the HS4h, but it was higher than that in the non-heated C4h group (P<0.05). In Experiment 2 (12 replicates), each matured oocyte was injected with IP(3) (0.5mM) and the Ca(2+) transient was recorded. The peak [Ca(2+)](i) in the C4h group was still the lowest among all groups (P<0.05). Total Ca(2+) release in HS2h appeared the highest among all treatments, and it was significantly higher than that in HS1h and C4h groups (P<0.05). In order to clarify the effect of incubation time in vitro (Experiment 3), matured oocytes were cultured at 39 degrees C for 0, 2 and 4h prior to treatment with thimerosal or injected with IP(3) (three replicates). The Ca(2+) release of matured oocytes declined with the prolonged culture (P<0.05). Finally, the development of HS-oocytes was evaluated after parthenogenetic activation (Experiment 4, three replicates), and the proportion of embryos developing to the blastocysts were lower (P<0.05) in the HS groups (31+/-7% to 33+/-1%) than in the control groups (52+/-11% to 56+/-9%). We conclude that HS alters the Ca(2+)-releasing ability of matured pig oocytes, and that heat-shocked oocytes with greater Ca(2+) release incur a low developmental competence after parthenogenetic activation.     

Shape change of human erythrocytes induced by phosphatidylcholine and lysophosphatidylcholine species with various acyl chain lengths

Intact human erythrocytes were treated, under non-haemolytic conditions at 37 degrees C, with synthetic phosphatidylcholine which has homologous, saturated acyl chains of 8-18 even-numbered carbon atoms (C8-C18-PC) or with lysophosphatidylcholine which has a saturated acyl chain of 8-18 carbon atoms (C8-C18-lysoPC). The C8-C14-PC and C12-C18-lysoPC species were rapidly incorporated into the erythrocytes and induced a shape change of the crenation (echinocyte formation) type. The site of the incorporation was found to be most probably on the outer leaflet of the membrane lipid bilayer. The extent of the shape change was dependent on the amount of each lipid incorporated. When the same amount of a PC or lysoPC species was incorporated into the membrane, about the same extent of crenation was induced, independent of acyl chain length. However, C16-PC, C18-PC, C8-lysoPC and C10-lysoPC, which were not incorporated into the erythrocytes, did not induce any shape change. It is therefore suggested that the hydrophobic moiety of these amphiphilic lipids may greatly contribute to their transfer from the outer medium into the erythrocyte membrane, but do not influence so much the perturbation of the membrane lipid bilayer which may be responsible for induction of the shape change.     

Diet-Induced Changes in AChE Activity after Long-Term Exposure

In the present study we investigated a potential mechanism by which high sugar (HS) and high fat (HF) diets could affect acetylcholinesterase (AChE) activity. The treatment with HS and HF diet was done for six months on male and female rats. The results showed decreased hippocampal AChE activity in male and females receiving HS and HF diets (HS 24% and 36%; HF 38% and 32%, males and females, respectively; P < 0.05). The activity in the cerebral cortex was reduced in males (49 and 40%) and females (19 and 17%) (P < 0.05) on HS and HF diets, respectively. In the hypothalamus AChE activity was decreased on HS diet in males (46%) and female (25%) (P < 0.05) and also on HF diet in males (34%) and females (21%) (P < 0.05). However, in the cerebellum no changes in AChE activity were observed. These results indicate that HS and HF diets produced mainly inhibition in acetylcholine degradation. It probably indicates a chronic alteration induced by these diets on the cholinergic system.     

Solubilization of Membrane-Bound Acetyicholinesterase by a Phosphatidylinositol-Specific Phospholipase C

Phosphatidylinositol-specific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ. The solubilized AChE migrates as a single peak of sedimentation coefficient 7.0S upon sucrose gradient centrifugation, corresponding to a subunit dimer. The catalytic subunit polypeptide of AChE is the only polypeptide detectably solubilized by PIPLC. This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K+ depolarization. PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes. The AChE released from rat and human erythrocytes by PIPLC migrates as a approximately 7S species on sucrose gradients, corresponding to a catalytic subunit dimer. PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species. Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum.     

Changes in Presynaptic Release of Acetylcholine During Development of Tolerance to the Anticholinesterase, DFP

The rat myenteric plexus was used as a peripheral model for studying muscarinic modulation of acetylcholine (ACh) release from presynaptic muscarinic neurons during development of tolerance to the anticholinesterase agent, diisopropylfluorophosphate (DFP). DFP in arachis oil was administered subcutaneously to intact animals according to both acute and chronic regimens, with arachis oil injections serving as controls. Post-mortem analyses showed that the mean AChE activity level in whole brain was reduced under all DFP conditions to 18.0 +/- 1.4% when compared with the control level. After 10 days of DFP treatment, the AChE level was 22.3 +/- 2.1% of control in the myenteric plexus. There were no significant differences among the treatment groups in resting ACh release. Release evoked by electrical stimulation (difference between stimulated and resting release) in the absence of atropine, i.e., "basal rate," for strips taken at various times after a single injection of DFP did not differ from that for strips from animals receiving arachis oil only. However, basal release for strips from chronically treated subjects was significantly greater than that of controls (p less than 10(-3), although not different from each other. Analysis of variance (ANOVA) for repeated measures showed that there existed a highly significant atropine dependency in strips from all treatments when they were stimulated in concentrations of atropine from 10(-9) to 10(-5) M (p less than 10(-10). Further analyses established that the increases in rates of evoked ACh release as concentrations of atropine increased were similar for strips from chronically treated DFP and arachis oil animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Indium-111 oxine labeled erythrocytes: Cellular distribution and efflux kinetics of the label

Indium-111 oxine labeled erythrocytes are useful in scintigraphic studies of splenic function because of the high yield of γ-photons [172(90%) and 247(94%) keV] of indium-111. However, the effects of indium-111 oxine on the structural and functional integrity of erythrocytes which might influence their reticulo-endothelial (RE) sequestration are unknown. We examined the morphology of human and rat indium-111 labeled erythrocytes by SEM, the distribution of the label within the cell by analysis of the membrane and cytosol (hemoglobin solution) and the kinetics of efflux of indium-111 from erythrocytes incubated at 37 °C in plasma or physiological buffer. Indium-111 oxine labeled red cells retain their discocytic morphology and the cell indices, and density characteristics on phthalate ester are similar to those of the control cells. The efficiency of labeling may be as high as 97%. Human or rat erythrocyte membranes retain 33 and 41% of indium-111, and the cytosol contains 67 and 59%, respectively. About 98% of the indium-111 is bound to the membrane proteins and 1% to the lipid bilayer. Efflux of indium-111 from cells in autologous plasma showed a multiphasic release resulting in about 4–5% release of the label in 2 h and 11.5% in 20 h. Cells in PBS showed 1–5% release of the label during the incubation period. These findings suggest that indium-111 oxine labeling of erythrocytes does not grossly alter the structural and deformability integrity of the cells to induce selective RE sequestration, unless the cells have been damaged prior to or during the labeling procedure, or the spleen is hyperactive.     

Phosphatidylinositol is involved in the attachment of tailed asymmetric acetylcholinesterase to neuronal membranes

1. We analyzed the mode of attachment of 16 S tailed acetylcholinesterase (AChE; EC 3.1.1.7) to rat superior cervical ganglion (SCG) neuronal membranes. Using extractions by high-salt (HS) and nonionic detergent (Triton X-100), we found two pools of 16 S AChE. 2. The detergent-extracted (DE) 16 S AChE was tightly bound to membranes through detergent-sensitive, high-salt insensitive interactions and was distinct from high-salt-soluble 16 S AChE. The detergent-extracted (DE) 16 S AChE constituted a significant proportion of about one-third of the total 16 S AChE. 3. Treatment of the neuronal membranes by a phosphatidylinositol-specific phospholipase C (PIPLC) resulted in the release of some, but not all DE 16 S AChE, indicating that a significant amount of the neuronal DE 16 S AChE, about one-third, is anchored to membranes through a phosphatidylinositol containing residue. Thus, a covalent association of a glycolipid and catalytic or structural AChE polypeptidic chains occurs not only for dimeric AChE but also for the asymmetric species of AChE. 4. The complex polymorphism of AChE is due not only to different globular or asymmetric associations of catalytic and structural subunits but also to the alternative existence of a transmembrane domain or a glycolipid membrane anchor.     

Rapid analysis of glycolipid anchors in amphiphilic dimers of acetylcholinesterases

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.     

A model-dependent approach to correlate accelerated with real-time release from biodegradable microspheres

The purpose of this study was to determine the feasibility of applying accelerated in vitro release testing to correlate or predict long-term in vitro release of leuprolide poly(lactideco-glycolide) microspheres. Peptide release was studied using a dialysis technique at 37°C and at elevated temperatures (50°C–60°C) in 0.1 M phosphate buffered saline (PBS) pH 7.4 and 0.1 M acetate buffer pH 4.0. The data were analyzed using a modification, of the Weibull equation. Peptide release was temperature dependent and complete within 30 days at 37°C and 3 to 5 days at the elevated temperatures. In vitro release profiles at the elevated temperatures correlated well with release at 37°C. The shapes of the release profiles at all temperatures were similar. Using the modified Weibull equation, an increase in temperature was characterized by an increase in the model parameter, α, a scaling factor for the apparent rate constant. Complete release at 37°C was shortened from ∼30 days to 5 days at 50°C, 3.5 days at 55°C, 2.25 days at 60°C in PBS pH 7.4, and 3 days at 50°C in acetate buffer pH 4.0. Values for the model parameter β indicated that the shape of the release profiles at 55°C in PBS pH 7.4 (2.740) and 50°C in 0.1 M acetate buffer pH 4.0 (2.711) were similar to that at 37°C (2.577). The E_a for hydration and erosion were determined to be 42.3 and 19.4 kcal/mol, respectively. Polymer degradation was also temperature dependent and had an E_a of 31.6 kcal/mol. Short-term in vitro release studies offer the possibility of correlation with long-term release, thereby reducing the time and expense associated with longterm studies. Accelerated release methodology could be useful in the prediction of long-term release from extended release microsphere dosage forms and may serve as a quality control tool for the release of clinical or commercial batches. Selected for the 2005 AAPS Outstanding Graduate Student Research Award in Pharmaceutical Technologies Sponsored by Solvay Pharmaceuticals.     

Glycolipid-anchored acetylcholinesterases from rabbit lymphocytes and erythrocytes differ in their sensitivity to phosphatidylinositol-specific phospholipase C.

The type of membrane association of acetylcholinesterase (AChE, EC 3.1.1.7) was studied in rabbit lymphocytes and erythrocytes. In both cases, the unique AChE molecular form was an amphiphilic dimer (referred to as G2a) anchored in the membrane by a glycosylphosphatidylinositol. In lymphocytes, G2a AChE was directly converted into its hydrophilic G2h counterpart by a treatment with Bacillus thuringiensis phosphatidylinositol-phospholipase C (PI-PLC, EC 3.1.4.10). In erythrocytes, AChE was resistant to PI-PLC but was rendered sensitive by a prior deacylation with alkaline hydroxylamine. This observation suggests that, as previously reported for human erythrocyte AChE, an acylation of the inositol ring in the glycolipid anchor of rabbit erythrocyte AChE (that does not occur in lymphocytes) prevents the cleavage.     

Amphiphile-induced antihaemolysis is not causally related to shape changes and vesiculation.

A wide variety of structurally different antihaemolytic amphiphiles were tested for their ability to induce exovesiculation (acetylcholinesterase (AChE) release, transmission electron microscopic (TEM) studies), endovesiculation (fluorescein isothiocyanate conjugated dextran (FITC-dextran) internalization, TEM studies) and shape changes in human erythrocytes at concentrations where they exert maximum protection against hypotonic haemolysis. The results show that vesiculation is a common phenomenon induced by amphiphiles in erythrocytes. Sphero-echinocytogenic amphiphiles induced exovesiculation, whereas stomatocytogenic amphiphiles induced endovesiculation. The antihaemolytic potency of the amphiphiles was not related to their ability to induce exo- or endovesiculation, or to the type or extent of shape changes induced, and it could not be ascribed to any molecular feature of the amphiphiles or to their charge. It is proposed that amphiphiles, when intercalated into the lipid bilayer of the membrane, rapidly induce rearrangements within the bilayer and that these rearrangements are associated with an increase in the permeability of the membrane; it is suggested that a rapid efflux of ions decreases the difference in osmotic pressure between cell interior and hypotonic buffer, thereby protecting cells from being lysed.     

Suppression of high-pressure-induced hemolysis of human erythrocytes by preincubation at 49 degrees C.

When human erythrocytes were preincubated at 37-52 degrees C under atmospheric pressure before exposure to a pressure of 200 MPa at 37 degrees C, the value of hemolysis was constant (about 43%) up to 45 degrees C but became minimal at 49 degrees C. The results from anti-spectrin antibody-entrapped red ghosts, spectrin-free vesicles, and N-(1-pyrenyl)iodoacetamide-labeled ghosts suggest that the denaturation of spectrin is associated with such behavior of hemolysis at 49 degrees C. The vesicles released at 200 MPa by 49 degrees C-preincubated erythrocytes were smaller than those released by the treatment at 49 degrees C or 200 MPa alone. The size of vesicles released at 200 MPa was independent of preincubation temperature up to 45 degrees C, and the vesicles released from 49 degrees C-preincubated erythrocytes became smaller with increasing pressure up to 200 MPa. Thus, hemolysis and vesiculation under high pressure are greatly affected by the conformation of spectrin before compression. Since spectrin remains intact up to 45 degrees C, the compression of erythrocytes at 200 MPa induces structural changes of spectrin followed by the release of large vesicles and hemolysis. On the other hand, in erythrocytes that are undergoing vesiculation due to spectrin denaturation at 49 degrees C, compression produces smaller vesicles, so that the hemolysis is suppressed.     

Dynamic association of band 3 with triton shells in human erythrocyte ghosts

To test a possibility that free band 3 and ankyrin-linked band 3 are exchanged in situ, band 3 was labeled with 125I, using intact red blood cells and lactoperoxidase. The cytoplasmic surface of this labeled band 3 was considered to be intact. When Triton shells were incubated with Triton supernatants prepared from 125I-labeled intact erythrocytes at 37 degrees C in the presence of Mg-ATP under isotonic conditions, the incorporation of free 125I-labeled band 3 to shells was observed. This incorporation was affected by the presence of Triton X-100 in the incubation mixture, and significantly decreased when the content of Triton X-100 was less than 0.04% (v/v). On the other hand, ankyrin-linked 125I-labeled band 3 was released when shells prepared from 125I-labeled intact erythrocytes were incubated with the Triton supernatants at 37 degrees C under the same condition as when free 125I-labeled band 3 incorporation was observed. These results strongly suggest that free and ankyrin-linked band 3 exchanged with each other in the presence of Triton X-100. A water-soluble 43 kDa fragment of band 3 inhibited the incorporation of free 125I-labeled band 3 to the shells and also inhibited the Mg-ATP-dependent shape change of ghosts in the absence of Triton X-100. Both of these inhibitory effects remained, even after 10 min of heat treatment at 100 degrees C, but drastically decreased by treatment with trypsin. Our results strongly suggest that a dynamic exchange of the free band 3 for ankyrin-linked band 3 may occur in intact erythrocytes, and it may even contribute to the shape change of erythrocytes.     

A spin-label study of membrane proteins and internal microviscosity of erythrocytes in hereditary spherocytosis

Electron spin resonance (ESR) spectra of erythrocyte membranes of patients with hereditary spherocytosis (HS) and of healthy controls labeled with a maleimide spin label did not differ significantly both before and after prolonged incubation at 37 degrees C. It suggests that the different behavior of spin-labeled HS erythrocyte membranes upon incubation at a higher temperature reported previously is due indeed to structural abnormalities of HS red cell membranes and not to alterations in their proteolytic activity. Measurements of the rotational correlation time of Tempamine spin probe demonstrated a significant elevation of internal microviscosity of erythrocytes in HS, more pronounced in non-splenectomized patients.     

The acetylcholinesterase (AChE) inhibitor and anti-Alzheimer drug donepezil interacts with human erythrocytes

Donepezil is used to treat symptomatically the Alzheimer's disease (AD). This drug is a specific inhibitor of the enzyme acetylcholinesterase (AChE), whose main physiological function is to hydrolyze the neurotransmitter acetylcholine. The main objective of this work was to study the effect of donepezil on human erythrocytes as AChE is present in its membrane. For this purpose, human erythrocytes and molecular model of its membrane built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) were used. The latter correspond to classes of phospholipids present in the outer and inner monolayers of the human erythrocyte membrane, respectively. Our experimental evidences obtained from X-ray diffraction and differential scanning calorimetry (DSC) analysis indicated that donepezil was capable of interacting with both phospholipids. Fluorescence spectroscopy results showed a moderate increase in the fluidity of the hydrophobic tails of DMPC and isolated unsealed human erythrocyte membranes (IUM). On the other hand, results by scanning electron microscopy (SEM) and optical defocusing microscopy (DM) showed that the drug changed the normal biconcave shape of the erythrocytes inducing the formation of stomatocytes (cup-shaped cells). This effect was explained by the incorporation of donepezil molecules into the erythrocyte membrane and interactions with AChE.     

Purification and properties of hydrophilic dimers of acetylcholinesterase from mouse erythrocytes

Differences in the glycosylation of acetylcholinesterase (AChE) subunits which form the dimers of mouse erythrocyte and a suitable procedure to purify the enzyme by affinity chromatography in edrophonium-Sepharose are described. AChE was extracted ( approximately 80%) from erythrocytes with Triton X-100 and sedimentation analyses showed the existence of amphiphilic AChE dimers in the extract. The AChE dimers were converted into monomers by reducing the disulfide bond which links the enzyme subunits. Lectin interaction studies revealed that most of the dimers were bound by concanavalin A (Con A) (90-95%), Lens culinaris agglutinin (LCA) (90-95%), and wheat germ (Triticum vulgaris) agglutinin (WGA) (70-75%), and a small fraction by Ricinus communis agglutinin (RCA(120)) (25-30%). The lower level of binding of the AChE monomers with WGA (55-60%), and especially with RCA (10-15%), with respect to the dimers, reflected heterogeneity in the sugar composition of the glycans linked to each AChE subunit in dimers. Forty per cent of the amphiphilic AChE dimers lost the glycosylphosphatidylinositol (GPI) and, therefore, were converted into hydrophilic forms, by incubation with phosphatidylinositol-specific phospholipase C (PIPLC), which permitted their separation from the amphiphilic variants in octyl-Sepharose. Only the hydrophilic dimers, either isolated or mixed with the amphiphilic forms, were bound by edrophonium-Sepharose, which allowed their purification (4800-fold) with a specific activity of 7700 U/mg protein. The identification of a single protein band of 66 kDa in gel electrophoresis demonstrates that the procedure can be used for the purification of GPI-anchored AChE, providing that the attached glycolipid domain is susceptible to PIPLC.