A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion

A small hydrophobic domain in isolated human erythrocyte acetylcholinesterase is responsible for the interaction of this enzyme with detergent micelles and the aggregation of the enzyme on removal of detergent. Papain has been shown to cleave this hydrophobic domain and to generate a fully active hydrophilic enzyme that shows no tendency to interact with detergents or to aggregate [Dutta-Choudhury, T.A., & Rosenberry, T.L. (1984) J. Biol. Chem. 259, 5653-5660]. We report here that the intact enzyme could be reconstituted into phospholipid liposomes while the papain-disaggregated enzyme showed no capacity for reconstitution. More than 80% of the enzyme reconstituted into small liposomes could be released by papain digestion as the hydrophilic form. Papain was less effective in releasing the enzyme from large liposomes that were probably multilamellar. In a novel application of affinity chromatography on acridinium resin, enzyme reconstituted into small liposomes in the presence of excess phospholipid was purified to a level of 1 enzyme molecule per 4000 phospholipid molecules, a ratio expected if each enzyme molecule was associated with a small, unilamellar liposome. Subunits in the hydrophilic enzyme form released from reconstituted liposomes by papain digestion showed a mass decrease of about 2 kilodaltons relative to the intact subunits according to acrylamide gel electrophoresis in sodium dodecyl sulfate, a difference similar to that observed previously following papain digestion of the soluble enzyme aggregates. The data were consistent with the hypothesis that the same hydrophobic domain in the enzyme is responsible for the interaction of the enzyme with detergent micelles, the aggregation of the enzyme in the absence of detergent, and the incorporation of the enzyme into reconstituted phospholipid membranes.     

Amphiphile dependency of the monomeric and dimeric forms of acetylcholinesterase from human erythrocyte membrane

Human erythrocyte membrane-bound acetylcholinesterase was converted to a monomeric species by treatment of ghosts with 2-mercaptoethanol and iodoacetic acid. After solubilization with Triton X-100, the reduced and alkylated enzyme was partially purified by affinity chromatography and separated from residual dimeric enzyme by sucrose density gradient centrifugation in a zonal rotor. Monomeric and dimeric acetylcholinesterase showed full enzymatic activity in presence of Triton X-100 whereas in the absence of detergent, activity was decreased to approx. 20% and 15%, respectively. Preformed egg phosphatidylcholine vesicles fully sustained activity of the monomeric species whereas the dimer was only 80% active. The results suggest that a dimeric structure is not required for manifestation of amphiphile dependency of membrane-bound acetylcholinesterase from human erythrocytes. Furthermore, monomeric enzyme appears to be more easily inserted into phospholipid bilayers than the dimeric species.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

Purification of detergent-solubilized form and membrane-binding domain of rat γ-glutamyltransferase by immuno-affinity and hydrophobic chromatography

A new method to purify papain- or detergent-solubilized form (papain or detergent form) of γ-glutamyltransferase from rat hepatomas as well as from rat kidney by immuno-affinity column chromatography is presented. The antibody-column was prepared by coupling the anti-kidney papain form antibody, which had been purified by using a kidney papain form-Sepharose column, to CNBr-activated Sepharose 4B. The enzyme bound to the antibody-column was eluted with 0.04 M NH₄OH. By this method, detergent forms were purified 300 and 1600-fold in approx. 50% yields from rat kidney and rat ascites hepatoma AH 13, respectively, and the papain form was also purified 16 000-fold in a similar yield from primary hepatoma which has a very low activity of this enzyme. Preparations thus obtained apparently did not contain any peptide other than heavy and light subunit peptides of this enzyme on SDS-polyacrylamide gel electrophoresis. The detergent form of kidney enzyme was preferentially adsorbed to a hydrophobic column of aminooctyl-Sepharose, while the papain form was not, suggesting that the detergent form might be adsorbed to the column through hydrophobic interaction of the membrane-binding domain. The domain peptide was also purified by the hydropholic column after release from the detergent form by papain treatment. The molecular weight of the peptide was estimated to be about 16 000 on SDS-polyacrylamide gel electrophoresis. On double immunodiffusion, the domain peptide reacted with anti-detergent form antibody but not with anti-papain form antibody. The domain-specific antibody was also purified from the anti-detergent form antibody.     

Visualization of collagenase-sensitive acetylcholinesterase in isolated cardiomyocytes and in heart tissue

Summary Previous studies have indicated that the asymmetric form of acetylcholinesterase (collagen-tailed) is localized in the basal lamina of the neuromuscular junction of skeletal muscle. The present study shows localization of the asymmetric acetylcholinesterase in the heart of the rat. Antiserum to 14+18 S acetylcholinesterase of the electric eel was raised in rabbits. The purified antibody did not react with collagen type I or laminin. Collagenase reduced the immunoreactivity of the enzyme with the purified antibody. Isolated cardiomyocytes and frozen sections of the heart were stained for acetylcholinesterase with the antibody. Diffuse immunofluorescence appeared over the surface of the cardiomyocytes. In the frozen sections, the immunofluorescence was most intense at the cell boundaries. These data suggest that collagenase-sensitive acetylcholinesterase in the heart is present in the myocytes and occurs in the vicinity of the basal lamina.Abbreviations AChE acetylcholinesterase - BSA bovine serum albumin - PBS phosphate-buffered saline - DME Dulbecco's Modified Eagle Medium     

A monomeric form of human erythrocyte membrane acetylcholinesterase

Purified detergent solubilized dimeric human erythrocyte acetylcholinesterase (6.3 S form) was converted to a stable monomeric 3.9 S species when treated with 2-mercaptoethanol and iodoacetic acid. More than 60% of the enzymatic activity were recovered after this treatment. A decreased susceptibility to reduction and alkylation was observed with purified, detergent depleted acetylcholinesterase aggregates. When erythrocyte membranes (ghosts) were subjected to the same treatment, acetylcholinesterase could subsequently be solubilized as monomeric 3.9 S form and and more than 90% of the activity were recovered. Monomeric acetylcholinesterase was less reactive towards antibodies raised against (dimeric) human erythrocyte membrane acetylcholinesterase and towards antibodies against human erythrocyte membranes. The results suggest that acetylcholinesterase is present as dimeric species in human erythrocyte membranes despite the fact that fully active monomers can be obtained.     

In vitro maintenance of nematode parasites assayed by acetylcholinesterase and allergen secretion.

A comparison was made of the ability of Nippostrongylus brasiliensis and Necator americanus to synthesize and secrete acetylcholinesterase when they were maintained in different in vitro culture media. The amount of allergen released by N. brasiliensis was also studied. The adult and fourth stage larval stages (but not the infective larvae) of Necator and adult N. brasiliensis secreted from their anterior glands up to 40 times as much acetylcholinesterase as they contained at the outset of culture. In contrast, allergen, which is less easy to quantitate, was secreted into the same media at about one-third the rate of secretion of acetylcholinesterase. Acetylcholinesterase was synthesized and released by both worms in media containing protein and the enzyme did not lose activity when kept for several days at 37 C. The secretion of this enzyme by nematodes kept in culture provides a simple, sensitive, rapid, and quantitative assay for measuring the ability of these nematodes to synthesize and secrete antigens in culture.     

The hydrophobic character of the membrane-bound phosphodiesterase from Dictyostelium discoideum

A phosphodiesterase activity is shown to copurify with the plasma membrane fraction prepared by the two-phase partition method. The enrichment in phosphodiesterase parallels that of alkaline phosphatase, which is thought to be a typical membranous enzyme. Up to 66% of the phosphodiesterase activity can be solubilized by a treatment with 0.2% Triton X-100. Higher doses were ineffective in solubilizing more activity. Analysis by native gel electrophoresis showed that an activity extracted by 2 M NaCl migrated at the same position as ‘soluble’ phosphodiesterase of cytosolic or extracellular origin. In contrast, the Triton-solubilized enzyme had an apparently higher molecular weight. When subjected to charge shift electrophoresis on agarose gels in the presence of an ionic detergent, the Triton-solubilized phosphodiesterase displayed a hydrophobic character. This behaviour contrasts with that of ‘soluble’ phosphodiesterases, the electrophoretic mobility of which is unaffected by the presence of an anionic detergent. The hydrophobic character of the membranous enzyme was lost after gentle hydrolysis by papain.     

Complex Effects of Papain on Function and Inhibitor Sensitivity of the Red Cell Anion Exchanger AE1 Suggest the Presence of Different Transport Subsites

Band 3 (AE1), the anion exchanger of the human erythrocyte membrane, mediates not only fluxes of small hydrophilic anions (e.g., chloride, oxalate), but also the flip-flop of long-chain amphiphilic anions (e.g., dodecylsulfate). Treatment of erythrocytes with papain, long known to inhibit the transport of the former type of anions, accelerates the transport of the latter type. In an attempt to elucidate the basis of these opposite responses to papain, several small amphiphilic arylalkyl sulfonates and -sulfates were tested for the response of their transport, via AE1, to papain. Although all these probes are most likely transported by a flux and not by flip-flop, their transport was inhibited by papain only in some cases, but accelerated in others. Different responses to papain therefore most likely do not reflect differences between transport by flux or by flip. The transports of different species of anions also differed considerably in the changes of their sensitivity, to noncovalent and some covalent inhibitors, brought about by papain treatment. While oxalate transport remained as sensitive as in native cells, transports of small amphiphilic anions lost their sensitivity to a major extent, regardless of the inhibition or acceleration of their transport by papain. The results are discussed in the light of present concepts of the structural organisation of AE1, and interpreted in terms of a model of different transport subsites for different species of anions in this transporter. Received: 20 June 2000/Revised: 1 November 2000     

The effect of exposure of acetylcholinesterase to 2,450-MHz microwave radiation

The effect of 2,450-MHz pulsed microwave radiation on the enzyme activity of membrane-free acetylcholinesterase was studied while the enzyme was in the microwave field. We found no significant effect of microwave radiation on enzyme activity using a wide variety of power densities, pulse widths, repetition rates, and duty cycles. This suggests that simple, direct modification by microwave energy of acetylcholinesterase structure and enzymic activity is not related to microwave alteration of acetylcholinesterase central nervous system levels.     

Inactivation of acetylcholinesterase by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride

The neurotoxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) has been shown to reversibly inhibit the activity of acetylcholinesterase. The inactivation of the enzyme was detected by monitoring the accumulation of yellow color produced from the reaction between thiocholine and dithiobisnitrobenzoate ion. The kinetic parameter, K _m for the substrate (acetylthiocholine), was found to be 0.216 mM and K _i for MPTP inactivation of acetylcholinesterase was found to be 2.14 mM. The inactivation of enzyme by MPTP was found to be dose-dependent. It was found that MPTP is neither a substrate of AChE nor the time-dependent inactivator. The studies of reaction kinetics indicate the inactivation of AChE to be a linear mixed-type inhibition. The dilution assays indicate that MPTP is a reversible inhibitor for AChE. These data suggest that once MPTP enters the basal ganglia of the brain, it can inactivate the acetylcholinesterase enzyme and thereby increase the acetylcholine level in the basal ganglia of brain, leading to potential cell dysfunction. It appears that the nigrostriatal toxicity by MPTP leading to Parkinson's disease-like syndrome may, in part, be mediated via the acetylcholinesterase inactivation.     

Electrical excitability of artificial enzyme membranes: III. Hysteresis and oscillations observed with immobilized acetylcholinesterase membranes

Experimental evidence for memory and oscillations in artificial acetylcholinesterase membranes is presented. When acetylcholine is injected on one side of an artificial proteinic membrane bearing acetylcholinesterase, a potential difference is recorded as a function of time. The steady-state potential due to the enzyme activity for increasing and decreasing substrate concentrations exhibits a hysteresis loop. The non-linearity of the enzyme reaction coupled with the diffusion constraints cause also some instabilities, such as oscillations of the membrane potential.     

Selective radiolabeling and isolation of the hydrophobic membrane-binding domain of human erythrocyte acetylcholinesterase

The hydrophobic, membrane-binding domain of purified human erythrocyte acetylcholinesterase was labeled with the photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. The radiolabel was incorporated when the enzyme was prepared in detergent-free aggregates, in detergent micelles, or in phospholipid liposomes, but the highest percentage of labeling occurred in the detergent-free aggregates. Papain digestion of the enzyme released the hydrophobic domain, and polyacrylamide gel electrophoresis in sodium dodecyl sulfate or gel exclusion chromatography demonstrated that the label was localized exclusively in the cleaved hydrophobic domain fragment. This fragment was purified in a three-step procedure. Digestion was conducted with papain attached to Sepharose CL-4B, and the supernatant was adsorbed to acridinium affinity resin to remove the hydrophilic enzyme fragment. The nonretained fragment associated with Triton X-100 micelles was then chromatographed on Sepharose CL-6B, and finally detergent was removed by chromatography on Sephadex LH-60 in an ethanol-formic acid solvent. The fragment exhibited an apparent molecular weight of 3100 on the Sephadex LH-60 column when compared with peptide standards. However, amino acid analysis of the purified fragment revealed only 1 mol each of histidine and glycine per mole of fragment in contrast to the 25-30 mole of amino acids expected on the basis of the molecular weight estimate. This result suggests a novel non-amino acid structure for the hydrophobic domain of human erythrocyte acetylcholinesterase.     

不同蕨类植物提取物乙酰胆碱酯酶抑制活性的评价

为对采自浙江天目山的40种蕨类植物乙醇提取物进行乙酰胆碱酯酶抑制活性研究。本文采用乙酰胆碱酯酶抑制活性筛选模型。结果表明：在供试质量浓度在1mg／mL时,40种植物的乙醇提取物均具有明显的乙酰胆碱酯酶抑制活性,其中密羽贯众、野雉尾金粉蕨和狗脊等13种蕨类植物表现出较强的抑制活性,抑制率均大于90％,其余大部分植物的抑制率均大于60％。而在供试质量浓度在0．025mg／mL时,只有少数植物表现出较高的乙酰胆碱酯酶抑制活性,其中狭顶鳞毛蕨的抑制率为62．63％±3．72％,蜈松草的抑制率为48．01％±2．87％,其余大部分植物的抑制活性均小于40％,甚至有些植物提取物不仅对乙酰胆碱酯酶没有抑制活性,反而表现出一定的增强作用。     

Expression of recombinant human acetylcholinesterase in transgenic tomato plants

Enzyme therapy for the prevention and treatment of organophosphate poisoning depends on the availability of large amounts of cholinesterases. Transgenic plants are being evaluated for their efficiency and cost-effectiveness as a system for the bioproduction of therapeutically valuable proteins. Here we report production of a recombinant isoform of human acetylcholinesterase in transgenic tomato plants. Active and stable acetylcholinesterase, which retains the kinetic characteristics of the human enzyme, accumulated in tomato plants. High levels of specific activity were registered in leaves (up to 25 nmol min(-1) mg protein(-1)) and fruits (up to 250 nmol min(-1) mg protein(-1)).     

Interaction of human red cell membrane acetylcholinesterase with phospholipids

Liposomes of phospholipids fully sustain the enzyme activity of the amphiphile-dependent dimers of human erythrocyte membrane acetylcholinesterase; no head group specificity exists. Diacylglycerides, glycerophosphorylcholine, or free fatty acids do not sustain the catalytic activity. It could be shown that the dimeric acetylcholinesterase with an exposed hydrophobic region can penetrate the lipid bilayer of liposomes and thus becomes stabilized by the surrounding phospholipid molecules.     

Monoclonal Antibodies to Rabbit Brain Acetylcholinesterase: Selective Enzyme Inhibition, Differential Affinity for Enzyme Forms, and Cross-Reactivity with Other Mammalian Cholinesterases

Eleven unique monoclonal IgG antibodies were raised against rabbit brain acetylcholinesterase (AChE, EC 3.1.1.7), purified to electrophoretic homogeneity by a two-step procedure involving immunoaffinity chromatography. The apparent dissociation constants of these antibodies for rabbit AChE ranged from about 10 nM to more than 100 nM (assuming one binding site per catalytic subunit). Species cross-reactivity was investigated with crude brain extracts from rabbit, rat, mouse cat, guinea pig, and human. One antibody bound rabbit AChE exclusively; most bound AChE from three or four species; two bound enzyme from all species tested. Identical, moderate affinity for rat and mouse brain AChE was displayed by two antibodies; two others were able to distinguish between these similar antigens. Nine of the antibodies had lowered affinity for AChE in the presence of 1 M NaCl, but two were salt resistant. Analysis of mutual interferences in AChE binding suggested that certain of the antibodies were competing for nearby epitopes on the AChE surface. One antibody was a potent AChE inhibitor (IC50 = 10(-8) M), blocking up to 90% of the enzyme activity. Most of the antibodies were less able to bind the readily soluble AChE of detergent-free brain extracts than the AChE which required detergent for solubilization. The extreme case, an antibody that was unable to recognize nearly half of the "soluble" AChE, was suspected of lacking affinity for the hydrophilic enzyme form.     

Sugar hydrolases of the infant rat intestine and their arrangement on the brush border membrane

Lactase and maltase, the predominant sugar hydrolases associated with the intestinal brush border membrane of the suckling rat, were purified essentially free of the other to near homogeneity (lactase at specific activity 23, maltase at specific activity 58), and their specific physicochemical properties determined. Antisera prepared to each showed by immunodiffusion a single common precipitin line with pure enzyme and solubilized proteins of the brush border membrane. Brush border membranes were purified 26–35-fold from infant rat intestine. Membranes prepared from 10-day-old rats contained 32% protein, 43% lipid and 25% carbohydrate with lactase and maltase estimated to comprise in excess of 10% and 2%, respectively, of the membrane protein.Immunotitration curves of lactase and maltase showed equivalent antibody binding by the membrane-bound and free enzyme forms. Furthermore, antibody binding to one enzyme did not affect the immunotitration curve or the extractability (by papain or Triton X-100) of the other membrane-bound enzyme. It was concluded that the lactase and maltase molecules are attached singly on the external membrane surface in a spatially independent manner with their antigenic sites as freely available to antibody binding as exhibited by their papain-solubilized counterparts.     

Properties of the leak permeability induced by a cytotoxic protein from Pseudomonas aeruginosa (PACT) in rat erythrocytes and black lipid membranes

A cytotoxic protein, isolated from Pseudomonas aeruginosa (PACT), was tested on red blood cells of rats and on black lipid membranes for changes of membrane permeability. In rat erythrocytes PACT induces lysis indicative of the formation of a leak permeable to monovalent ions. The dose response curve for the PACT-induced hemolysis demonstrates that the rate of lysis as well as the fraction of lytic cells increases with increasing toxin concentration. Furthermore, the leak pathway discriminates hydrophilic non-electrolytes according to their molecular weight. The findings indicate formation by PACT of a pore with an apparent radius of about 1.2 nm. In pure lipid membranes PACT forms hydrophilic pathways with moderate selectivity for small cations over small anions. The presence of cholesterol is a prerequisite for the occurrence of these PACT-induced permeability changes.     

不同温度下锐劲特对受马拉硫磷抑制的麦穗鱼AChE活性恢复的影响

研究了１５～１７℃、２０～２２℃下,０．０１ｍｇ／Ｌ锐劲特对被１ｍｇ／Ｌ马拉硫磷抑制的麦穗鱼（Ｐｓｅｕｄｏｒａｓｂｏｒａ ｐａｒｖａ）脑乙酰胆碱酯酶（ＡＣｈＥ）活性恢复的影响。结果表明：在１５～１７℃,经４８ｈ抑制,ＡＣｈＥ被抑制了４０％,恢复１４４ｈ后,处理组与无锐劲特对照都恢复到了空白对照的８０％以上。在此温度下锐劲特对ＡＣｈＥ活性恢复的影响不明显;而在２０～２２℃下,经相同的处理时间,ＡＣｈＥ被抑制了７０％,恢复２４０ｈ后,处理组与无锐劲特对照都只恢复到了对照的７０％。其间于１４４、１９２ｈ,ＡＣｈＥ活性差异达显著水平（Ｐ〈０．０５）,说明了在此温度下锐劲特对ＡＣｈＥ活性恢复的影响。文中还讨论了恢复研究的意义。