Acetylcholinesterase from Apis mellifera head. Evidence for amphiphilic and hydrophilic forms characterized by Triton X-114 phase separation.

The polymorphism of bee acetylcholinesterase was studied by sucrose-gradient-sedimentation analysis and non-denaturing electrophoretic analysis of fresh extracts. Lubrol-containing extracts exhibited only one form, which sedimented at 5 S when analysed on high-salt Lubrol-containing gradients and 6 S when analysed on low-salt Lubrol-containing gradients. The 5 S/6 S form aggregated upon removal of the detergent when sedimented on detergent-free gradients and was recovered in the detergent phase after Triton X-114 phase separation. Thus the 5 S/6 S enzyme corresponds to an amphiphilic acetylcholinesterase form. In detergent-free extracts three forms, whose apparent sedimentation coefficients are 14 S, 11 S and 7 S, were observed when sedimentations were performed on detergent-free gradients. Sedimentation analyses on detergent-containing gradients showed only a 5 S peak in high-salt detergent-free extracts and a 6 S peak, with a shoulder at about 7 S, in low-salt detergent-free extracts. Electrophoretic analysis in the presence of detergent demonstrated that the 14 S and 11 S peaks corresponded to aggregates of the 5 S/6 S form, whereas the 7 S peak corresponded to a hydrophilic acetylcholinesterase form which was recovered in the aqueous phase following Triton X-114 phase separation. The 5 S/6 S amphiphilic form could be converted into a 7.1 S hydrophilic form by phosphatidylinositol-specific phospholipase C digestion.     

Molecular Forms of Acetylcholinesterase in Bovine Caudate Nucleus and Superior Cervical Ganglion: Solubility Properties and Hydrophobic Character

Abstract: In the present paper, we report an analysis of acetylcholinesterase molecular forms in the bovine caudate nucleus and superior cervical ganglion. We show that: (1) The superior cervical ganglion contains a significant proportion (～ 15%) of collagen-tailed forms (mostly A₁₂ and A₈), but these molecules are found only as traces (ca. 0.002%) in the caudate nucleus, even in favorable extraction conditions (i.e., in the presence of 1 m -NaCl, 5 mm -EDTA, 1% Triton X-100). (2) The bulk of acetylcholinesterase corresponds to globular forms, mostly the tetrameric G₄ and the monomeric G₁ forms, with a smaller proportion of the dimeric G₂ form. (3) The tetrameric enzyme exists as a minor soluble component (G^S₄) that does not interact with Triton X-100, and a major hydrophobic component (G^H₄) that is partially solubilized in the absence of detergent in the caudate nucleus, but not in the superior cervical ganglion. (4) The monomeric G₁ form presents a marked hydrophobic character, as indicated by its interaction with Triton X-100, although it may be solubilized in large part in the absence of detergent in both tissues. (5) The detergentsolubilized forms aggregate upon removal of detergent. This property disappears after partial purification of G₄) that does not interact with Triton X-100, and a major hydrophobic component (G^H₄, but is restored upon addition of an inactivated crude extract, indicating that it is attributable to interactions with other hydrophobic components. (6) The proportions of molecular forms solubilized in detergent-free buffers vary with the ionic composition of the medium. Repeated extractions of caudate nucleus in Tris-HCl buffer produce a larger overall yield of G₁ form (e.g., 40%) than appears in a single quantitative detergent solubilization (<15%). This G₁ form apparently derives in part from a pool of G^H₄ form. (7) However, detergents that allow a quantitative solubilization of acetylcholinesterase yield the same proportions of forms (about 85% G₄) independently of the ionic conditions. (8) Modifications of the molecular forms occur spontaneously during purification, or storage of the crude aqueous ex-tracts, in a manner that depends on the ionic conditions. In Tris-HCl buffer, G₁ is converted into a well-defined 7.5S form. In Ringer, polydisperse components are formed. The effects observed in Ringer cannot be reproduced by addition of 5 mm -Ca_2- to the Tris buffer either during or after extraction. (9) Proteases, such as pronase, convert the hydrophobic forms into molecules that do not appear to interact with Triton X-100, and do not aggregate in its absence. These results raise fundamental questions regarding the status of acetylcholinesterase in situ, the structure and interactions of its molecular forms. They are discussed with reference to previous publications.     

Properties of acetylcholinesterase and non-specific cholinesterase in rat superior cervical ganglion and plasma

Amphiphile dependency, solubility in aqueous solutions, and sensitivity to proteolysis of acetylcholinesterase (AChE) and nonspecific cholinesterase (nsChE) in the rat superior cervical ganglion were studied and compared to properties of soluble plasma cholinesterases. Ganglion AChE shows strong amphiphile dependency: an amphyphilic substance must be present in the homogenizing medium in order to obtain maximal apparent enzyme activity. Apparent activity of AChE solubilized in Ringer's solution was also increased after subsequent addition of a detergent. The 4 S molecular form, predominant in this extract (corresponding to the fastest electrophoretic band), is very sensitive to papain proteolysis but can be protected by a detergent. This molecular form therefore carries an important hydrophobic domain and is probably membrane bound in situ. The 10 S form of ganglionic AChE, extracted in Ringer's solution, is probably a soluble enzyme since, like soluble plasma enzymes, it is not amphiphile dependent and is rather resistant to proteolysis. Ganglion nsChE is more water soluble, less amphiphile dependent and more protease resistant than AChE.     

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.     

Characterization of acetylcholinesterase molecular forms in slow and fast muscle of rat

Multiple molecular forms of acetylcholinesterase (AChE EC 3.1.1.7) from fast and slow muscle of rat were examined by velocity sedimentation. The fast extensor digitorum longus muscle (EDL) hydrolyzed acetylcholine at a rate of 110 mumol/g wet weight/hr and possessed three molecular forms with apparent sedimentation coefficients of 4S, 10S, and 16S which contribute about 50, 35, and 15% of the AChE activity. The slow soleus muscle hydrolyzed acetylcholine at a rate of 55 mumol/g wet weight/hr and has a 4S, 10S, 12S, and 16S form which contribute 22, 18, 34, and 26% of AChE activity, respectively. A single band of AChE activity was observed when a 1M NaCl extract with CsCl (0.38 g/ml) was centrifuged to equilibrium. Peak AChE activity from EDL and SOL extracts were found at 1.29 g/ml. Resedimentation of peak activity from CsCl gradients resulted in all molecular forms previously found in both muscles. Addition of a protease inhibitor phenylmethylsulfonyl chloride did not change the pattern of distribution. The 4S form of both muscles was extracted with low ionic strength buffer while the 10S, 12S, and 16S forms required high ionic strength and detergent for efficient solubilization. All molecular forms of both muscles have an apparent Km of 2 x 10(-4) M, showed substrate inhibition, and were inhibited by BW284C51, a specific inhibitor of AChE. The difference between these muscles in regards to their AChE activity, as well as in the proportional distribution of molecular forms, may be correlated with sites of localization and differences in the contractile activity of these muscles.     

Characterization of acetylcholinesterase activity from Drosophila melanogaster

The acetylcholinesterase activity of the fruit fly, Drosophila melanogaster, was characterized biochemically. The activity is associated with a glycoprotein which is divided between a detergent-extractable membrane-bound fraction and a soluble fraction. The acetylcholinesterase activity is concentrated in the head of the insect. Through pharmacological methods, greater than 95% of the cholinesterase is judged to be true acetylcholinesterase, and not pseudocholinesterase. As expected for an acetylcholinesterase, the enzyme has a high affinity for acetylthiocholine and is inhibited by excess concentrations of acetylthiocholine. The soluble enzyme is found predominantly as a 7.8 S form; a smaller amount of an approximately 6 S form is also present, and a greater than or equal to 14 S form may exist. The detergent-solubilized acetylcholinesterase has a sedimentation coefficient of 7.5 S in the presence of detergent. The thermal inactivation rates for the soluble and the membrane bound enzymes are markedly different.     

Thermal stability of molecular forms of acetylcholinesterase from muscle microsomes

To establish if the predominant form of acetylcholinesterase in muscle microsomes (4.8S) corresponded to the monomeric or dimeric form of the enzyme we studied the sensitivity to heating of Triton X-100 solubilized extract and that of 4.8S, 10-11S and 13.5S species of the enzyme. Inactivation of soluble acetylcholinesterase began at 45-47 degrees C and was almost complete at 60 degrees C. Sedimentation analysis revealed that the partial loss of activity was due to inactivation of the 4.8S form, although by heating the 13.5S was converted into the 10S enzyme. Inactivation of the 4.8S form began at 45 degrees C, whereas the larger forms required higher temperature. The 4.8S component follows a time course of inactivation which could be fitted by a double exponential equation (when heated at 52 degrees C, almost 83% of the activity showed a short half-life). The 10-11S species was also inactivated following a two step process while the 13.5S enzyme was fairly stable at 52 degrees C. The results show that the lightest component behaves as a monomeric form of acetylcholinesterase.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.     

Interaction of estrogen receptor of calf uterus with a monoclonal antibody: probing of various molecular forms

A monoclonal antibody to estrogen receptor (JS34/32) is able to recognize, in the calf uterine cytosol, a protein (approximately 65 000 daltons) giving a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Two molecules of this antibody are able to simultaneously interact with the native 8S form of the receptor present in the calf uterine cytosol ("twin antibody" assay). This indicates the presence of two antigenic determinants on the "low-salt" 8S form of the receptor. This form of the receptor shows an increase in Mr from 345 000 to 665 000 after interaction with the soluble antibody. Dissociating agents that induce the dissociation of the 8S form to smaller forms also induce the dissociation of the two antigenic determinants. The 4S "high-salt" form of the estrogen receptor has one determinant per molecule, appearing to be the smallest form of the receptor not containing repetitive structures associated with the steroid binding site. The nuclear receptor also shows the presence of more than one antigenic determinant on its molecule.     

Molecular forms of acetylcholinesterase present in the white and grey matter of pig brain

Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100 gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove acetylcholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter.The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter.Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge.Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed.A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.     

Properties of a 5′-nucleotidase purified from mouse liver plasma membranes

1. Extraction of a mouse liver plasma-membrane fraction with a detergent buffer, N-dodecylsarcosinate-Tris buffer (sarcosyl-Tris buffer), solubilized 90% of the protein and 70% of the 5'-nucleotidase activity. 2. The proteins of the sarcosyl-Tris buffer extract were fractionated by a rate-zonal centrifugation in a sucrose-detergent gradient. The major protein peak sedimented ahead of phospholipids, which mainly remained in the overlay. Glycoproteins were separated ahead of the protein peak. 3. The 5'-nucleotidase activity peak was associated with 5% of the protein applied to the gradient, and contained relatively few protein bands. 4. The 5'-nucleotidase was purified further by gel filtration on Sepharose and Sephadex columns equilibrated with sarcosyl-Tris buffer, to give a single glycoprotein band on sodium dodecyl sulphate-polyacrylamide-gel electrophoresis. The purified enzyme was lipid-free. 5. Electrophoresis in polyacrylamide gels in sarcosyl-Tris buffers showed that the enzymic activity was coincident with the protein band. 6. The molecular weight suggested for the enzyme activity by gel filtration or centrifugation in sucrose gradients was 140000-150000. Sometimes, a minor enzyme peak of lower molecular weight was obtained. 7. Polyacrylamide-gel electrophoresis in sodium dodecyl sulphate indicated that as the polyacrylamide concentration was increased from 5 to 15%, the apparent molecular weight of the enzyme decreased from 130000 to 90000. 8. The evidence that 5'-nucleotidase is composed of two active and similar, if not identical, glycoprotein subunits and the role of detergent in effecting the separation of membrane proteins and glycoproteins are discussed. 9. Substrate requirements, pH optima and the nature of inhibition by an analogue of adenosine diphosphate are reported.     

Recombinant human acetylcholinesterase is secreted from transiently transfected 293 cells as a soluble globular enzyme

1. Coding sequences for the human acetylcholinesterase (HuAChE; EC 3.1.1.7) hydrophilic subunit were subcloned in an expression plasmid vector under the control of cytomegalovirus IE gene enhancer-promoter. The human embryonic kidney cell line 293, transiently transfected with this vector, expressed catalytically active acetylcholinesterase. 2. The recombinant gene product exhibits biochemical traits similar to native "true" acetylcholinesterase as manifested by characteristic substrate inhibition, a Km of 117 microM toward acetylthiocholine, and a high sensitivity to the specific acetylcholinesterase inhibitor BW284C51. 3. The transiently transfected 293 cells (100 mm dish) produce in 24 hr active enzyme capable of hydrolyzing 1500 nmol acetylthiocholine per min. Eighty percent of the enzymatic activity appears in the cell growth medium as soluble acetylcholinesterase; most of the cell associated activity is confined to the cytosolic fraction requiring neither detergent nor high salt for its solubilization. 4. The active secreted recombinant enzyme appears in the monomeric, dimeric, and tetrameric globular hydrophilic molecular forms. 5. In conclusion, the catalytic subunit expressed from the hydrophilic AChE cDNA species has the inherent potential to be secreted in the soluble globular form and to generate polymorphism through self-association.     

Isolation and Partial Characterization of a Cytochrome b Complex and Cytochrome Oxidase from Yeast Mitochondria

A cytochrome b complex and cytochrome oxidase have been purified 14- and 20-fold respectively from yeast submitochondrial particles by a simple procedure involving their spontaneous precipitation from a deoxycholate extract. The recovery of both proteins was almost quantitative. The specific heme contents were 11 and 8 nmoles/mg protein for the cytochrome b complex and cytochrome oxidase respectively and both were spectrally pure. Sodium dodecyl sulfate gel electrophoresis resolved the cytochrome b complex into seven distinct subunits with molecular weights 42, 000, 33, 000, 27, 500, 23, 000, 15, 500, 13, 000 and 10, 500. Cytochrome oxidase contained five bands with molecular weights 42, 000, 26, 500, 21, 000, 14, 000 and 10, 500. Much of the cytochrome b complex (and all of the cytochrome oxidase) could be resolubilized in aqueous buffer following precipitation from the deoxycholate extract. The fraction of the cytochrome b preparation which remained insoluble appeared identical to the soluble protein in terms of polypeptide composition but contained less phospholipid and bound detergent, suggesting that insolubility may result from interaction between hydrophobic regions otherwise occupied by amphiphiles. The soluble cytochrome b complex migrated as a single species upon analytical ultracentrifugation and column chromatography, and during electrophoresis on polyacrylamide gels. Triton X-100, urea, or bile salts, failed to dissociate the complex. These findings suggest that the subunits are tightly associated in situ.     

Intracellular distribution of molecular forms of acetylcholinesterase in rat brain and changes after diisopropylfluorophosphate treatment

The subcellular distribution of acetylcholinesterase activities was studied in the striatum and cerebellum of rat brain. The highest percentage of the enzyme activity was found in the crude synaptosomal (P₂) fraction, with striatum much higher than cerebellum. On sucrose density gradient centrifugation analyses all the particulate fractions (P₁, P₂, and P₃) showed a major peak of the 10 S form of acetylcholinesterase activity with very little activity of the 4 S form of the enzyme. The 10 S/4 S ratio was much higher in striatum than in cerebellum. In the soluble fraction (100,000g supernatant) the 10 S form was less than the 4 S form in the adult rat brain, but this was reversed in the 6-day-old rat brain. After diisopropylfluorophosphate administration the recovery of acetylcholinesterase molecular forms in various subcellular fractions differed at different recovery periods. These results indicate that the distribution of molecular forms of acetylcholinesterase in rat brain differs in various subcellular fractions, and also the pattern of distribution differs in different regions of the brain as well as in adult and developing brains.     

Molecular form of human lymphocyte membrane-bound acetylcholinesterase

The membrane-bound acetylcholinesterase (AchE) from human peripheral blood lymphocyte gives only one symmetrical peak on sucrose density gradient centrifugation in the presence of Triton X-100 detergent, with the calculated sedimentation coefficient of 6.5 S. However, this dimeric form of AchE was converted to a monomeric 3.8 S form when treated with 2-mercaptoethanol and iodoacetic acid. The results are consistent with studies which have shown by sodium dodecyl sulfate gel electrophoresis that the enzyme is built up of two identical monomers inter-linked by disulfide bond(s). Under reducing conditions, revealed a single species of 70,000 molecular weight, whereas under non-reducing conditions, another species of 140,000 molecular weight of the AchE was found. Polyacrylamide gel electrophoresis indicated a single band with AchE activity in the presence of Triton X-100. In contrast, in the absence of the same detergent multiple band pattern could be observed. These results suggest that membrane-bound AchE enzyme is present in homogenous dimeric form on human lymphocyte membrane.     

Cholinesterases from flounder muscle. Purification and characterization of glycosyl-phosphatidylinositol-anchored and collagen-tailed forms differing in substrate specificity

Flounder (Platichthys flesus) muscle contains two types of cholinesterases, that differ in molecular form and in substrate specificity. Both enzymes were purified by affinity chromatography. About 8% of cholinesterase activity could be attributed to collagen-tailed asymmetric acetylcholinesterase sedimenting at 17S, 13S and 9S, which showed catalytic properties of a true acetylcholinesterase. 92% of cholinesterase activity corresponded to an amphiphilic dimeric enzyme sedimenting at 6S in the presence of Triton X-100. Treatment with phospholipase C yielded a hydrophilic form and uncovered an epitope called the cross-reacting determinant, which is found in the hydrophilic form of a number of glycosyl-phosphatidylinositol-anchored proteins. This enzyme showed catalytic properties intermediate to those of acetylcholinesterase and butyrylcholinesterase. It hydrolyzed acetylthiocholine, propionylthiocholine, butyrylthiocholine and benzoylthiocholine. The Km and the maximal velocity decreased with the length and hydrophobicity of the acyl chain. At high substrate concentrations the enzyme was inhibited. The p(IC50) values for BW284C51 and ethopropazine were between those found for acetylcholinesterase and butylcholinesterase. For purified detergent-soluble cholinesterase a specific activity of 8000 IU/mg protein, a turnover number of 2.8 x 10(7) h-1, and 1 active site/subunit were determined.     

Bacterial phosphotransferase system. Solubilization and purification of the glucose-specific enzyme II from membranes of Salmonella typhimurium

The glucose-specific enzyme II (IIGlc) of the phosphoenolpyruvate-dependent phosphotransferase system of Salmonella typhimurium has been purified to homogeneity. Purification included the following steps: detergent solubilization of membranes in polydisperse octyloligooxyethylene, isoelectrofocusing, chromatofocusing, and either glycerol gradient centrifugation or gel filtration, all in the presence of the same detergent. Enzymatic activity was assayed as phosphoenolpyruvate-dependent phosphorylation of methyl-alpha-D-glucopyranoside. It could be measured after detergent dilution only and required the presence of phosphatidylglycerol in a sonicated suspension. An antiserum prepared against enzyme IIGlc specifically inhibited phosphorylation of methyl-alpha-D-glucopyranoside. In the solubilized state, purified enzyme IIGlc exists as a complex of molecular weight of 105,000 and a sedimentation coefficient of 3.8 S. In polyacrylamide gels in sodium dodecyl sulfate, it has an apparent molecular weight of about 40,000.     

Molecular structure of elongated forms of electric eel acetylcholinesterase.

Molecular forms of acetylcholinesterase extracted from fresh electric organ tissue of the electric eel are elongated structures in which a multi-subunit head is connected to a fibrous tail. The principal form, 18 S acetylcholinesterase, is of molecular weight approximately 1,050,000, contains about 12 catalytic subunits in its head, has a tail approximately 500 Å long, and aggregates reversibly at low ionic strength. Trypsin converts it to an 11 S globular tetramer devoid of the tail and lacking the capacity to aggregate in low-salt solutions.Amino acid analysis shows that elongated forms of acetylcholinesterase contain significant amounts of hydroxyproline and hydroxylysine, characteristic components of collagen, which are absent from 11 S acetylcholinesterase.Collagenase converts 18 S acetylcholinesterase to a 20 S form which no longer aggregates in low salt. Purified 20 S acetylcholinesterase has about half the hydroxyproline and hydroxylysine contents of the 18 S enzyme, and physicochemical measurements indicate the formation of a more symmetrical molecular structure without marked reduction in molecular weight.Sodium dodecyl sulfate/polyacrylamide gel electrophoresis without reducing agent shows that in 18 S acetylcholinesterase half the catalytic subunits are present as dimers linked by disulfide bonds. The remaining subunits migrate as larger molecular species which contain significant amounts of hydroxylysine, are specifically modified by collagenase and are converted to dimers and monomers by trypsin.Sodium dodecyl sulfate/acrylamide gel electrophoresis with reducing agent reveals, in 18 S acetylcholinesterase, two polypeptides of molecular weights 45,000 and 47,000 which are absent in the 11 S tetramer. They are readily digested by collagenase under conditions which do not affect the catalytic subunits, with concomitant formation of a new 30,000 polypeptide.The above data can be rationalized by a model in which 18 S acetylcholinestorase contains three subunit tetramers, each linked by disulfides to one strand of a collagen triple helix. Sodium dodecyl sulfate detaches those subunit dimers which are not covalently linked to the tail; trypsin attacks the distal portion of the collagen triple helix releasing discrete tetramers, and collagenase specifically attacks the triple helix near its midpoint, producing a shortened structure in which the residual tail still holds the tetramers together, but destroying the capacity for self-association at low ionic strength. This latter property may be related to the postulated role of the tail in anchoring acetylcholinesterase to the fibrillar matrix of the basement membrane.     

Solubilization of Collagen-Tailed Acetylcholinesterase from Chick Retina: Effect of Different Extraction Procedures

We have extracted acetylcholinesterase from young chick retinas by homogenization in different solutions combining high salt concentration, ionic and nonionic detergents, and EDTA, looking for an optimum procedure for the solubilization of collagen-tailed, asymmetric structural forms of the enzyme. High salt and EDTA seem to be the only necessary requirements for the solubilization of acetylcholinesterase as the A12 form (20S), and the presence of detergent in the homogenization medium does not significantly improve the yield of tailed enzyme. Extraction in the absence of detergent has the potential advantage of a threefold enrichment of tailed enzyme, because only about one-third of the total retinal acetylcholinesterase activity is solubilized. Divalent cations, especially Ca2+, seem to be involved in the attachment of the tailed enzyme to the retinal membranes, at the tail level. High salt-EDTA-extracted 20S acetylcholinesterase (without detergent) aggregates in the presence of exogenous Ca2+ and becomes "insoluble." However, the aggregated 20S acetylcholinesterase can be completely recovered and brought back into solution by further addition of EDTA. Besides, the aggregation can be prevented by the inclusion of Triton X-100 in the homogenization buffer or by adding the detergent concurrently with Ca2+. It is postulated that the acetylcholinesterase collagenous tail is coated by acidic lipid molecules hydrophobically bound to the tail protein so that Ca2+ ionic bridges would actually link these lipid molecules (and consequently the tail) to the membrane matrix. Removal of the lipid coat (e.g., by Triton X-100) produces tailed acetylcholinesterase molecules that no longer aggregate in the presence of Ca2+ and are fully accessible to collagenase digestion.     

Soluble form of acetylcholinesterase from rabbit enterocytes: comparison of its molecular properties with those of the plasma membrane species

A soluble form of acetylcholinesterase was shown to be present in rabbit enterocytes. The enzyme was obtained from a high-speed supernatant (105,000 X g centrifugation) after homogenization of intestinal mucosa without detergent. It was shown to possess no obvious hydrophobic character and could be classified as a low-salt-soluble (LSS) acetylcholinesterase. Sucrose gradient centrifugation revealed a single enzyme species with a sedimentation coefficient of 3.9 +/- 0.2S. By gel filtration performed in HPLC the enzyme was eluted as a protein corresponding to an Mr of 72,000 +/- 3,000. It could be precipitated with concanavalin A by affinoelectrophoresis, but the catalytic activity was not affected by the lectin. Our results are consistent with a G1 globular form for this soluble acetylcholinesterase which differs very clearly from detergent-soluble forms also found recently in the plasma membranes of rabbit enterocytes.