Distribution and origin of acetylcholinesterase activity in the capillaries of the brain.

Areas containing AChE-positive capillaries were mapped in the brain of the cat and the guinea pig. Regions with AChE-positive capillaries mostly also contain neuronal elements with AChE activity. Electron-microscopical cytochemistry revealed localization of AChE in basement membranes of endothelial cells and pericytes very often in continuity with activity of the extracellular space. Intraendothelial AChE activity was seen only in pinocytic vesicles. The vascular AChE is thought to be of neuronal origin since no cytochemical evidence has been obtained for a synthesis of this enzyme in endothelial or other non-neuronal cells in the CNS.     

Acetylcholinesterase in membrane fractions derived from sarcotubular system of skeletal muscle: presence of monomeric acetylcholinesterase in sarcoplasmic reticulum and transverse tubule membranes

Microsomes were isolated from white rabbit muscle and separated into several fractions by centrifugation in a discontinuous sucrose density gradient. Four membrane fractions were obtained namely surface membrane, light, intermediate and heavy sarcoplasmic reticulum. The origin of these microsomal vesicles was investigated by studying biochemical markers of sarcoplasmic reticulum and surface and T-tubular membranes. The transverse tubule derived membranes were further purified by using a discontinuous sucrose density gradient after loading contaminating light sarcoplasmic reticulum vesicles with calcium phosphate in the presence of ATP. All membrane preparations displayed acetylcholinesterase activity (AChE, EC 3.1.1.7), this being relatively more concentrated in T-tubule membranes than in those derived from sarcoplasmic reticulum. The membrane-bound AChE of unfractioned microsomes notably increased its activity by aging, treatment with detergents and low trypsin concentrations indicating that the enzyme is probably attached to the membrane in an occluded form, the unconstrained enzyme displaying higher activity than the vesicular acetylcholinesterase.Sedimentation analysis of Triton-solubilized AChE from different membrane fractions revealed enzymic multiple forms of 13.5S, 9–10S and 4.5–4.8S, the lightest form being the predominant one in all membrane preparations. Therefore, in both sarcoplasmic reticulum and T-tubule membrane the major component of AChE appears to be a membrane-bound component, probably a G₁ form.     

Distribution and origin of acetylcholinesterase activity in the capillaries of the brain

Summary Areas containing AChE-positive capillaries were mapped in the brain of the cat and the guinea pig. Regions with AChE-positive capillaries mostly also contain neuronal elements with AChE activity. Electron-microscopical cytochemistry revealed localization of AChE in basement membranes of endothelial cells and pericytes very often in continuity with activity of the extracellular space. Intraendothelial AChE activity was seen only in pinocytic vesicles. The vascular AChE is thought to be of neuronal origin since no cytochemical evidence has been obtained for a synthesis of this enzyme in endothelial or other non-neuronal cells in the CNS.Drs. H. Kaiya and L. Toth were recipients of research fellowships granted by the Max Planck Society     

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.     

Acetylcholinesterase activity in the adrenal chromaffin vesicles of Urodela

The presence of acetylcholinesterase (AChE) activity in the adrenal chromaffin cells of Necturus maculosus and Ambystoma maculatum (Amphibia, Urodela) has been demonstrated by cytochemical method at the electron microscope level. The enzymatic activity is localized in RER and perinuclear cisternae, on the plasma membrane and within the chromaffin vesicles, both in adrenaline (A) and noradrenaline (N) cells. Moreover N cells appear to be more reactive than A cells and Necturus more reactive than Ambystoma. The possible function of the AChE activity inside the vesicles is discussed as a mechanism of protons donor or as peptidasic activity acting on various peptides present in the vesicle.     

Acetylcholinesterase is orientated facing the cytoplasmic side in membranes derived from sarcoplasmic reticulum

(1) Microsomal membranes from white rabbit muscle enriched in sarcoplasmic reticulum (SR) were used to investigate the preferential localization of acetylcholinesterase (AChE) in these membranes. (2) Integrity and orientation of the vesicles was assessed by measuring the inulin-inaccessible space of the vesicles and its calcium-loading capacity. (3) Treatment of the membranes with diisopropyl phosphorofluoridate (DFP), an irreversible inhibitor which is free soluble in lipid, produced an almost complete inactivation of AChE. The inhibition was prevented in assays performed with the non-permeant reversible inhibitor BW 284c51 (BW). (4) Similar results were obtained if echothiophate iodide (ECHO), an irreversible and poorly permeant inhibitor, instead of DFP was used. (5) Sedimentation profiles of enzyme solubilized with Triton X-100 from membranes inhibited by DFP after protection with BW showed a minor reduction in the relative proportion of a 4.5 S (G1) form. (6) Treatment of intact or saponin-permeabilized membranes with concanavalin A (ConA) produced enzyme-lectin complexes. In both cases, most of the enzyme was recovered in the sedimented complexes after centrifugation of the Triton-solubilized membranes. (7) Incubation of intact membranes with the antibody AE1 led to the formation of immuno complexes. Sedimentation analyses of the molecular forms of AChE revealed a shift in the sedimentation coefficients, whether the antibody was added before or after solubilization of the enzyme. (8) These results firmly establish an external localization of AChE in SR, most of the protein backbone facing the cytoplasmic side of the membrane.     

Glycosylation of Acetylcholinesterase Forms in Microsomal Membranes from Normal and Dystrophic Lama2dy Mouse Muscle

Abstract: The distribution and glycosylation of acetylcholinesterase (AChE) forms in vesicles derived from sarcoplasmic reticulum of normal muscle (NMV) were investigated and compared with those from dystrophic muscle vesicles (DMV). AChE activity was similar in NMV and DMV. Most of the AChE in NMV and half in DMV were released with Triton X-100. Asymmetric (A₁₂) and globular hydrophilic and amphiphilic (G^H₄, G^A₄, G^A₂, and G^A₁) AChE species occurred in NMV and DMV, the lighter forms being predominant. The percentage of G^H₄ and G^A₄ decreased in DMV. A fraction of the AChE that could not be extracted with detergent was detached with collagenase. Most of the detergent-released A₁₂ AChE from NMV and nearly half in DMV failed to bind to Ricinus communis agglutinin (RCA-I). Conversely, the collagenase-detached isoforms bound to RCA, revealing that asymmetric AChE associated with internal membranes or basal lamina differed in glycosylation. Moreover, nearly half of G^A₄ AChE in DMV and a few in NMV bound to RCA. Most of the RCA-unreactive G^A₄ forms in NMV come from sarcolemma. The results indicate that dystrophy induces minor changes in the distribution and glycosylation of AChE forms in internal membranes of muscle.     

Ultrastructural evidence for dendritic release of acetylcholinesterase in the rat substantia nigra

Morphological evidence for dendritic secretion of acetylcholinesterase (AChE) in rat substantia nigra--a physiologically known phenomenon--was searched by means of a modified cytochemical method devised for fine localization of AChE activity at the electron microscopic level. DAB precipitate was observed in cluster of small vesicles in contact with the plasma membrane and in the extracellular space in the vicinity of the vesicles. Single coated or uncoated large vesicles filled with stained material were found in the cytoplasm of the dendrites at distance from or in contact with the plasma membrane. Immunoperoxidase staining with specific anti-serum against rat AChE gave similar localization of AChE. These results suggest that AChE is released from the dendrites of the nigral neurons by a process of vesicular exocytosis and captured by endocytosis. The relation of this process to a putative release from the smooth endoplasmic reticulum remains to be elucidated.     

Biochemical and cytochemical evidence indicates that coated vesicles in chick embryo myotubes contain newly synthesized acetylcholinesterase

We have isolated highly purified coated vesicles from 17-d-old chick embryo skeletal muscle. These isolated coated vesicles contain acetylcholinesterase (AChE) in a latent, membrane-protected form as demonstrated enzymatically and morphologically using the Karnovsky and Roots histochemical procedure (J. Histochem. Cytochem., 1964, 12:219- 221). By the use of appropriate inhibitors the cholinesterase activity can be shown to be specific for acetylcholine. It also can be concluded that most of the AChE represents soluble enzyme since it is rendered soluble by repeated freeze-thaw cycles. To determine the origin of the coated vesicle-associated AChE, we have isolated coated vesicles from cultured chick embryo myotubes which have been treated with diisopropylfluorophosphate, an essentially irreversible inhibitor of both intra- and extracellular AChE, and have been allowed to recover for 3 h. This time is not enough to allow any newly synthesized AChE to be secreted. These coated vesicles also contain predominantly soluble AChE. These data are compatible with the hypothesis that coated vesicles are important intermediates in the intracellular transport of newly synthesized AChE.     

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.     

Dot-blot test for identification of insecticide-resistant acetylcholinesterase in single insects

A test was developed to detect the presence of insecticide-resistant acetylcholinesterase (AChE) in single insects based on the quasipermanent binding of proteins onto blotting membranes. The method is simple, sensitive, requires inexpensive equipment, and produces a permanent record of results. AChE activity is revealed by the Karnovsky & Roots staining technique in the presence of propoxur, or after exposure of the membrane to paraoxon and rinsing with water. We chose insecticide concentrations that inhibited the sensitive AChE while allowing detectable residual activity of the resistant AChE to remain. By comparing the staining of insecticide-treated and control membranes, susceptible and resistant genotypes for the AChE gene could be distinguished in laboratory strains of mosquitoes (Culex spp. and Anopheles albimanus Wiedemann) and the house fly (Musca domestica L.). Resistant AChE from mosquitoes was less susceptible both to propoxur and paraoxon than the corresponding sensitive AChE, whereas resistant AChE from house fly was less susceptible mainly to paraoxon. The technique worked well for mosquito adults and house fly heads but not for mosquito larvae. Blotted AChE did not show detectable loss of activity during storage of the membranes for 3 wk at 25 degrees C. Storage is an important asset of the technique because transportation of live insect material to the laboratory may not be necessary.     

Biochemical and cytochemical localization of ATPases on the membranes of the electrocyte of Electrophorus electricus

Summary The localization of (Na⁺-K⁺) ATPase in the intact electrocyte of the electric organ of Electrophorus electricus (L.) and its subcellular fractions was investigated by biochemical and cytochemical methods. The distribution of AChE activity in the subcellular fractions was also comparatively analysed with this enzyme serving as a marker of the innervated membranes of the electrocyte. After application of cytochemical method of Farquhar and Palade to glutaraldehyde-fixed tissue, reaction was observed only at the membranes of vesicles localized at the periphery of the electrocyte. Previously fixed electrocytes, incubated in Ernst's medium showed reaction only at the vesicles whereas in unfixed tissue reaction also appeared at other membranes (surface and invaginations) of the anterior and posterior faces. This reaction was significantly inhibited in the presence of ouabain or in the absence of K⁺. Inhibition of Na⁺-K⁺-ATPase by glutaraldehyde fixation was also confirmed by biochemical analysis.This investigation has been supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico, Conselho de Ensino e Pesquisa da UFRJ and FINEP (FNDCT-375/CT)     

Electron microscopic histochemistry of acetylcholinesterase of rat brain by Karnovsky's method

Summary Karnovsky's electron microscopic acetylcholinesterase method was successfully applied to rat brain fixed by vascular perfusion with either 2% glutataldehyde or 4% formaldehyde. 2% glutaraldehyde showed better fine structure but worse preservation of the enzyme than 4% formaldehyde.In the neuropil of the caudate nucleus, locus coeruleus and dorsal nucleus of the vagus, AChE activity was most intensely demonstrated on the plasma membranes of preterminal axons and somewhat less strongly on those of axon terminals and contacting dendritic branches. The axoplasm and synaptic vesicles were usually negative, while the cytoplasm and neurotubules of the dendritic branches showed some activity. In the nodule and uvula of the cerebellum moderate activity was exhibited on the synaptic contacts between the mossy fiber endings and granule cell dendrites. In the hypothalamus and other autonomic regions the characteristic coexistence of AChE and granulated vesicles of axon terminals could be demonstrated.In the perikaryon of positive nerve cells, AChE was observed strongly in the cytoplasm, disseminated irregularly or attached to the endoplasmic reticulum, while it was absent in the mitochondria and lysosomal dense bodies.     

CHARACTERIZATION OF MEMBRANES OBTAINED FROM ELECTRIC ORGAN OF THE ELECTRIC EEL BY SUCROSE GRADIENT FRACTIONATION AND BY MICRODISSECTION

Abstract— Two membrane fractions were obtained from electric organ tissue of the electric eel by sucrose gradient centrifugation of tissue homogenates. Electron microscopic examination showed that both fractions contained mainly vesicular structures (microsacs). Both the light and heavy fractions had a-bungarotoxin-binding capacity and Na⁺-K⁺ ATPase activity, while only the light fraction had AChE activity. The polypeptide patterns of vesicles derived from both the light and heavy fractions were examined by SDS-polyacrylamide gel electrophoresis and found to be very similar. The ratio of protein to phospholipid in the light vesicles was much lower than in the heavy vesicles, but the relative amounts of individual phospholipids in the two fractions were similar. A marked difference in the permeability of the light and heavy vesicles was observed by measuring efflux of both [¹⁴C]sucrose and ²²Na⁺, and also by monitoring volume changes induced by changing the osmotic strength of the medium. All three methods showed the heavy vesicles to be much more permeable than the light ones. Only the light vesicles displayed increased sodium efflux in the presence of carbamylcholine. The AChE in the light fraction does not appear to be membrane-bound, but is rather a soluble enzyme, detached from the membrane during homogenization, which migrates on the gradient similarly to that of the light vesicles. This is supported by the fact that the bulk of the AChE is readily removed by washing the vesicles. Moreover, under the conditions employed in our sucrose gradient separations,‘native’14 S + 18 S AChE exists in the form of aggregates which migrate very similarly to the major peak of AChE activity of tissue homogenates. Separated innervated and non-innervated surfaces of isolated electroplax were obtained by microdissection. α-Bungarotoxin-binding capacity was observed only in the innervated membrane. About 80% of the AChE was in the innervated membrane, and about 70% of the Na⁺-K⁺ ATPase in the non-innervated membrane. The data presented indicate that the light and heavy vesicle fractions separated by sucrose gradient centrifugation are not derived exclusively from the innervated and non-innervated membranes respectively, as previously suggested by others, but contain membrane fragments from both sides of the electroplax. The separation of two populations on sucrose gradients may be explained both by the differences in permeability and in protein to phospholipid ratios.     

Acetylcholinesterase activity at the synapses of presynaptic boutons with presumed alpha-motoneurons in chicken ventral horn. Light- and electron-microscopic studies

Acetylcholinesterase (AChE) activity at the synapses of presynaptic boutons on presumed alpha-motoneurons in the chicken ventral horn was studied histochemically at the light- and electron-microscope levels. At the light-microscope level, many dot-like AChE-active sites were observed on the soma and dendrites of presumed alpha-motoneurons. On electron microscopy, reaction products for AChE activity were observed mainly in the synaptic clefts of the four kinds of presynaptic boutons: (1) S type boutons, (2) boutons containing small, spherical, dense cored vesicles (diameter range, 60-105 nm) and spherical, clear vesicles, (3) boutons containing medium-sized, spherical, dense cored vesicles (65-115 nm) and spherical, clear vesicles, and (4) boutons containing large, spherical, dense cored vesicles (80-130 nm) and spherical, clear vesicles. In the light of previous physiological and biochemical studies, the present results suggest the possibility that each of these presynaptic boutons which are AChE-active in their synaptic clefts may contain acetylcholine, substance P, or enkephalins which acts as a neurotransmitter or modulator.     

INCREASE IN THE APPARENT AChE ACTIVITY IN THE MOUSE DIAPHRAGM INDUCED BY PROTEOLYTIC TREATMENT

Abstract— Isolated endplate regions from the mouse diaphragm were treated with different agents before or after homogenization in order to solubilize junctional AChE and study the effect of solubilization on its apparent activity. Total AChE activity (solubilized + nonsolubilized) of samples treated with collagenase or papain before homogenization was nearly twice as high as in control samples. If collagenase was added after homogenization no increase in apparent activity was observed although in both cases about 70–80% of AChE activity was solubilized. The access of ACh to the membrane-bound enzyme is probably not a limiting factor in the AChE assay as is the case in the electric organ homogenates. Both 1 m -NaCl and Triton X-100 were quite ineffective as solubilizers when applied before homogenization and had an insignificant effect on the apparent AChE activity.
The increase in apparent AChE activity cannot be explained either by a de novo synthesis or by the change in kinetic properties of different species of AChE, or by the release of AChE possibly sequestrated in the membrane vesicles. The possibility is discussed that a part of junctional AChE is inactivated at the beginning of homogenization while it can be preserved by previous solubilization, or that proteolytic treatment may activate some 'silent' AChE sites in motor endplates.
However, the mere fact that the difference does exist suggests that all AChE activity present in intact motor endplates may not be measurable after homogenization.     

Ultrastructural localization of acetylcholinesterase in the synganglion of the tick,Dermacentor variabilis (Say)

Summary Light- and electron-microscopic enzyme cytochemistry was used to localize acetylcholinesterase (AChE) activity in the synganglion (brain) of the tick Dermacentor variabilis. High AChE activity was observed throughout the neuropil as well as adjacent to most neuronal perikarya. Intracellular activity was not observed by light microscopy. By electron microscopy, reaction product was localized at the plasma membrane of glia and neurons. Enzyme activity was not associated with the olfactory globuli neurons. In other types of neurons, small amounts of reaction product were observed in the Golgi apparatus and nuclear envelope. Large neurosecretory neurons contained activity that appeared to be associated with deep invaginations of the plasma membrane as well as intracellular membranes. AChE activity was also associated with processes of both neurons and glia. In most peripheral nerves AChE activity was associated with virtually all axons. Clearly then, AChE is associated with glia and non-cholinergic neurons as well as with presumed cholinergic neurons. The widespread localization and large amounts of AChE in the tick brain exceeds that reported for other invertebrates and vertebrates. As has been suggested for other animals, AChE in the tick brain may have functions in addition to its known role in cholinergic neurotransmission.     

Cytochemistry of cytochrome oxidase in the cytoplasmic and intracytoplasmic membranes of Azotobacter vinelandii

Vegetative cells of Azotobacter vinelandii contain a system of intracytoplasmic membranes in the form of numerous internal vesicles. The three-dimensional morphology of these internal vesicles was established by an examination of stereopair electron micrographs of negatively stained cells. The vesicles assumed a variety of forms ranging from nearly spherical units to short, curved tubules. These structures were found at the periphery of the cytoplasm, subjacent to the cytoplasmic membrane. Large flattened cisternae were also present in some cells. The amount of intracytoplasmic membrane varied widely even among individual cells from the same culture. The total surface area of the intracytoplasmic membranes was greater than that of the cytoplasmic membrane in many cells. To assess the possible association of cytochrome oxidase activity with the intracytoplasmic membranes, enzyme localization experiments were conducted with the cytochemical substrate 3,3'-diaminobenzidine. The results showed that a cyanide-sensitive cytochrome oxidase activity is located at the intracytoplasmic membrane. The quantity of cytochrome oxidase activity present in the internal membranes is probably less than that present in the cytoplasmic membrane.     

LOCALIZATION AND PROPERTIES OF THE CHOLINESTERASE IN CRUSTACEAN MUSCLE

Cholinesterase (ChE) activity is present in crustacean muscle extracts. However, since acetylcholine (ACh) is not a neuromuscular transmitter in these animals, the role and exact localization of ChE was unknown. The histochemical localization of the enzyme was studied in whole muscle and in the sarcoplasmic reticulum fraction of the extract, 50-µm frozen sections of glutaraldehyde-fixed crayfish tail flexor muscle were incubated with acetylthiocholine (ATC) as substrate, and examined under the electron microscope. After some modifications in published techniques, dense deposits were found associated with the sarcolemma, sarcolemmal invaginations, and transverse tubules. No deposits were found in 10^-4 M eserine, or if butyrylthiocholine (BTC) was substituted for ATC. The vesicles in the sarcoplasmic reticulum fraction which demonstrate the activity must represent minced bits of these membranes. Using a spectrophotometric method, the kinetics of the crustacean muscle enzyme was compared to the acetylcholinesterase (AChE) on mammalian red blood cells and in the lobster ventral nerve cord. Surprisingly, and contrary to previous reports, the crustacean muscle enzyme did not demonstrate substrate inhibition. While a number of similarities to AChE were found, this lack of substrate inhibition makes questionable an unequivocal similarity with classical AChE.     

TRANSPORT OF CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN THE RAT SCIATIC NERVE: A BIOCHEMICAL AND ELECTRON HISTOCHEMICAL STUDY

The transport of acetylcholinesterase (AChE) and choline acetyltransferase (ChAc) were investigated by biochemical and histochemical methods. After ligature of one of the sciatic nerves of the rat for varying times—4, 14, 20 and 44 h—the normal levels and the accumulation of AChE and ChAc activities were investigated. It can be inferred from the results that there is a rapid accumulation of AChE activity just proximal to the ligature, while the increase in ChAc activity is less pronounced. Distal to the ligature the level of AChE is above the control value whereas, in contrast to this, the ChAc activity is significantly decreased. Histochemical demonstration of the two enzymes indicates that they are present in the cholinergic axons. The reaction end-product produced by AChE occurs within vesicles and neurotubules, while the endproduct due to ChAc appears to be free in the axoplasm, bound to neurofilaments and on the outer surface of vesicles and tubules.