Characterization of triton X-100-solubilized prostaglandin E binding protein of rat liver plasma membranes.

Rat liver plasma membranes bind prostaglandins E1 and E2 (PGE) with high affinity and specificity. We have solubilized plasma membranes, prelabeled with radioactive PGE1, in water solutions of Triton X-100. We sedimented this material into sucrose density gradient containing H2O and D2O. From numerical integration of the sedimentation equation, taking explicitly into account the density and viscosity gradients present during the centrifugation, we have determined a value of s20,w = 5.6 to 5.7 X 10(-13) s and a partial specific volume, v = 0.80 to 0.81 cm3/g, for the PGE binding protein-Triton X-100 composed of 60% (w/w) protein and 40% (w/w) detergent. Gel filtration in water solutions of Triton X-100 gives a Stokes radius of 53 A for the complex. These data imply a molecular weight of 105,000 for the detergent-free binding protein and a frictional ratio of 1.3 for the complex. If the detergent is bound to the protein in a monolayer, about 40% of the PGE binding protein's surface would be covered with detergent. The procedures used in the analysis of the sedimentation behavior of the PGE binding protein-detergent complex, when coupled with a gel filtration measurement of the Stokes radius, allow valid determination of the size, shape, and extent of detergent binding of a wide variety of membrane proteins, even when they are present as minor components of complex mixtures.     

Triton X-100 induced dissociation of beef heart cytochrome c oxidase into monomers

Purified beef heart cytochrome c oxidase, when solubilized with at least 5 mg of Triton X-100/mg of protein, was found to be a monodisperse complex containing 180 molecules of bound Triton X-100 with a protein molecular weight of 200 000, a Stokes radius of 66-72 A, and an s(0)20,w = 8.70 S. These values were determined by measurement of the protein molecular weight by sedimentation equilibrium in the presence of D2O, evaluation of the sedimentation coefficient, S(0)20,w, by sedimentation velocity with correction for its dependence upon the concentration of protein and detergent, and measurement of the effective radius by calibrated Sephacryl S-300 gel chromatography. The monomeric complex was judged to be homogeneous and monodisperse since the effective mass of the complex was independent of the protein concentration throughout the sedimentation equilibrium cell and a single protein schlieren peak was observed during sedimentation velocity. These results are interpreted in terms of a fully active monomeric complex that exhibits typical biphasic cytochrome c kinetics and contains 2 heme a groups and stoichiometric amounts of the 12 subunits normally associated with cytochrome c oxidase. With lower concentrations of Triton X-100, cytochrome c oxidase dimers and higher aggregates can be present together with the monomeric complex. Monomers and dimers can be separated by sedimentation velocity but cannot be separated by Sephacryl S-300 gel filtration, probably because the size of the Triton X-100 solubilized dimer is not more than 20% larger than the Triton X-100 solubilized monomer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Convulsant/barbiturate activity on the soluble gamma-aminobutyric acid-benzodiazepine receptor complex

Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.     

The preparation of hydrophilic derivatives of band 3 protein by acylation of the human red blood cell membrane

In situ reaction of erythrocyte membranes with dicarboxylic anhydrides leads to solubilization of hydrophobic integral proteins. Removal of peripheral proteins and bulk lipid by appropriate sedimentation and dialysis steps yields hydrophilic band 3 protein derivatives. These acyl compounds display size heterogeneity upon gel filtration. A chromatographically homogeneous acyl band 3 protein is obtained if the acylation is conducted in the presence of detergent and the detergent subsequently removed. Hydrophilic acyl derivatives of band 3 protein can be subjected to conventional analytical techniques without the use of detergents.     

Solubilization and partial characterization of the tetrodotoxin binding component from nerve axons

The tetrodotoxin binding component from garfish olfactory nerve membranes has been solubilized using the nonionic detergent Triton X-100. Tetrodotoxin binds to the solubilized component with a dissociation constant K_D = 2.5 × 10^?9$\text{M}$ and under saturating conditions 1.95 × 10^?12 moles of tetrodotoxin are bound per milligram of solubilized protein. Upon solubilization the toxin binding component becomes much less stable towards heat, chemical modification and enzymatic degradation. Sucrose gradient velocity sedimentation yields an S value of 9.2 for the extracted binding component and from gel filtration data the binding component appears to be slightly larger than β-D-galactosidase.     

Identification and solubilization of atrial natriuretic factor receptors in human placenta

Specific high affinity 125I-atrial natriuretic factor binding sites have been identified in human placental membranes. Using the nonionic detergent, Triton X-100, these binding sites were quantitatively solubilized and retained binding activity. In the solubilized preparation, the macromolecular component that binds atrial natriuretic factor is a 160,000 dalton protein as shown by covalently cross-linking it to 125I-atrial natriuretic factor with the bifunctional chemical crosslinker, disuccinimidyl suberate, followed by gel electrophoresis and autoradiography. On Sephadex G-200 gel filtration in the presence of detergent, the hormone-receptor complex elutes in the molecular weight range of 140,000. These observations suggest strongly that a 140- 160,000 dalton protein present in human placental membranes is the receptor for specific recognition of atrial natriuretic factor.     

Disassembly and characterization of the nuclear pore complex-lamina fraction from bovine liver nuclei

The nuclear pore complex-lamina (PCL), composed of nuclear pore structures attached to fibrous lamina, was isolated from bovine liver nuclei. We found that the highly aggregated PCL was disrupted and 75% of the constituent polypeptides could be solubilized by extraction for 1 h with 2% deoxycholate (DOC) and 3% 2-mercaptoethanol. While some differential solubilization was observed at lower detergent concentrations, all PCL proteins were solubilized equally at 2% DOC. The reducing agent was necessary to achieve maximum dispersal of the PCL and to prevent aggregation of the solubilized proteins. No tightly bound phospholipid or Triton X-100 could be detected in these preparations. Rapid removal of DOC, by dialysis or gel filtration, resulted in aggregation and precipitation of the PCL proteins, but the detergent could be removed by centrifugation through sucrose gradients. The sedimentation profiles indicated that the three major polypeptides, lamins A, B, and C, each sedimented as a single peak with a shoulder of more rapidly sedimenting material, possibly higher oligomeric forms. The sedimentation coefficient of lamins B and C, in the presence and absence of detergent, was 4.5 S. In the presence of DOC, lamin A had a sedimentation coefficient of 5.6 S, but this value was decreased to 4.1 S, when DOC was omitted from the gradient. These studies suggested that lamins B and C do not interact with or bind DOC, while lamin A may bind appreciable amounts of the detergent. The Stokes radii of lamins A, B, and C were found by gel filtration to be 75, 75, and 70 A, respectively. The molecular weights and frictional ratios estimated from the sedimentation and gel filtration data indicated that the lamins are dimeric, rod-shaped molecules.     

Solubilization and partial purification of hyaluronate synthetase from oligodendroglioma cells

Hyaluronate synthetase was solubilized with digitonin from crude membranes of mouse oligodendroglioma cells. Detergent extraction was carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline with an optimal digitonin to protein ratio (w/w) of 0.7-0.8. The solubilized synthetase was partially purified approximately 230-fold by gel filtration and ion-exchange chromatography. The solubilized enzyme displayed similar properties to membrane-bound enzyme: (a) it synthesized high molecular weight hyaluronate which eluted in the void volume of a Sepharose CL-2B column; (b) the apparent Km values obtained for UDP-GlcUA and UDP-GlcNAc were 50 and 100 microM, respectively; and (c) treatment of intact cells with hyaluronidase prior to extraction with digitonin resulted in a 3-fold increase in solubilized synthetase activity. Furthermore, gel filtration chromatography of the solubilized hyaluronidase-treated synthetase complex showed that it was smaller than the solubilized untreated synthetase complex, due to shorter nascent-bound hyaluronate. The solubilized synthetase was shown to be associated with hyaluronate in the form of a complex. Both hyaluronidase-treated and -untreated synthetase-hyaluronate complexes after solubilization were adsorbed by an affinity matrix using the hyaluronate binding domain of rat chondrosarcoma proteoglycan as ligand. This solubilized active enzyme preparation should allow the identification and characterization of the components of the hyaluronate-synthetase complex.     

Biophysical characterization of the purified alpha 1-adrenergic receptor and identification of the hormone binding subunit

The alpha 1-adrenergic receptor has been solubilized in active form from rat hepatic membranes with the nonionic detergent, digitonin, and purified by affinity and gel filtration chromatography to homogeneity with a specific activity of 14,400 pmol/mg of protein. The affinity chromatographic steps of the purification procedure were achieved by the use of a newly synthesized analog (2-[4(2-succinoyl)piperazin-1-yl]-4-amino-6,7-dimethoxyquinazoline, CP-57,609) of the highly selective alpha 1-adrenergic antagonist, prazosin, immobilized via an amide linkage to agarose. The resulting purified receptor bound [3H]prazosin and a variety of adrenergic agents with the specificity, stereoselectivity, and affinities equivalent to those observed with membrane-bound and solubilized receptor preparations. The purified receptor.digitonin complex had a Stokes radius of 49 A and a sedimentation coefficient (s20w) of 7.1, as determined by AcA-34 gel filtration chromatography and sucrose gradient density centrifugation, respectively. Based on these hydrodynamic parameters, the calculated molecular weight of the receptor.digitonin complex was estimated at 147,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis following the final purification step revealed a single band of protein at 59,000 daltons from which [3H]prazosin binding activity could be recovered after renaturation of the receptor protein. These findings indicate that the protein purified from rat hepatic membranes is the hormone binding component of the alpha 1-adrenergic receptor and that the receptor molecule most likely contains more than one Mr = 59,000 subunit.     

Solubilization, gel filtration and sedimentation behaviour of prolactin receptors from human ovarian tissue

Receptors for 125I-labelled human prolactin have been identified in the crude membrane fraction isolated from human ovarian tissue. The non-ionic detergent Triton X-100, has been used to solubilize the membrane fraction. The presence of the receptor in the detergent extract was demonstrated by gel filtration and sucrose density gradient centrifugation. The binding was time-temperature dependent, being maximal at 23 degrees C after 15 h of incubation. Large amounts of other peptide hormones did not inhibit the binding of 125I-labelled human prolactin. The binding Scatchard analysis demonstrated that the affinity of the soluble receptor (Ka 1.13 +/- 0.15 X 10(10) M-1) for the labelled hormone was slightly greater than that of the crude membrane fraction (Ka 0.91 +/- 0.12 X 10(10) M-1). The binding capacity of the solubilized receptor was also significantly greater than that seen in the particulate before solubilization. The apparent Stokes radius of the solubilized receptor was estimated to be 57 A and that the hormone-receptor complex 60 A. The sedimentation coefficient of the solubilized receptor was 7.0 +/- 0.1 s, whereas that of the hormone-receptor complex was 7.5 +/- 0.2 s.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with ¹²⁵I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of ¹²⁵I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed ¹²⁵I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Hydrodynamic properties of a thromboxane A2/prostaglandin H2 antagonist binding site solubilized from human platelets

The hydrodynamic properties of a binding site for the thromboxane A2/prostaglandin H2 receptor antagonist 9,11-dimethylmethano-11, 12-methano-16-(3-iodo-4-hydroxyphenyl)-13, 14-dihydro-13-aza-15 alpha beta-omega-tetranor-thromboxane A2 (I-PTA-OH) were determined in solubilized membrane proteins from human platelets using the detergent 3-[(3-cholamidopropyl)-dimethylammonio] 1-propane-sulfonate (CHAPS). Gel filtration revealed a Stokes radius of 5.25 +/- 0.37 nm (n=9). Molecular weight determined by gel filtration assuming a spherical protein was 180,000-220,000 Daltons. Sedimentation through sucrose or glycerol gradients revealed a sedimentation coefficient of 6.3 +/- 0.2 Svedberg units (n=5). The molecular weight calculated using the Stokes radius and sedimentation coefficient was 140,000 Daltons. The frictional ratio f/fo was 1.4, corresponding to an axial ratio of 7:1.     

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum.

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 X g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with an apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similarly, the free receptor also showed higher sedimentation profile with an apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI. U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the preformed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.     

Size characteristics of the solubilized saxitoxin receptor of the voltage-sensitive sodium channel from rat brain

The saxitoxin receptor of the voltage-sensitive sodium channel from rat brain was solubilized with Triton X-100 and stabilized with phosphatidylcholine. The size characteristics of the detergent . phospholipid . receptor complex were studied by gel filtration and sucrose gradient sedimentation in H2O and D2O. The complex has Stokes radius = 80 A, S20,W = 12 S, v = 0.82 ml/ g, and Mr = 601,000 +/- 48,000. Assuming v = 0.73 ml/g for the saxitoxin receptor protein, the mass of the complex consists of 47.4% detergent and phosphatidylcholine and 52.6% saxitoxin receptor protein with Mr = 316,000 +/- 63,000.     

Solubilization of Calcium Channel Antagonist Binding Sites from Rat Brain

Calcium antagonist binding sites were solubilized from rat brain membranes using the detergent 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulfonate (CHAPS). CHAPS-solubilized [3H]nitrendipine binding sites are saturable over a range of 0.05-4 nM and Scatchard analysis reveals a single, high-affinity (KD = 0.49 +/- 0.10 nM), low-capacity (Bmax = 56 +/- 4 fmol/mg of protein) binding site. Reversible ligand competition experiments using solubilized binding sites demonstrated appropriate pharmacologic specificity, with dihydropyridines (nifedipine = nitrendipine greater than Bay K 8644) completely displacing binding, verapamil partially displacing binding, and diltiazem enhancing binding, as previously described in membrane preparations. Lyophilized Crotalus atrox venom was purified by ion exchange chromatography followed by gel filtration to a single peptide band on sodium dodecyl sulfatepolyacrylamide gel electrophoresis. This fraction of molecular weight 60,000 competitively inhibits [3H]nitrendipine binding to both membrane and soluble preparations with an IC50 of 5 micrograms/ml. This polypeptide should serve as a useful ligand for future efforts in purifying the dihydropyridine calcium channel binding site in brain.     

Solubilization and molecular weight estimation of atrial natriuretic factor receptor from bovine adrenal cortex

Receptors for atrial natriuretic factor (ANF) have been solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate from bovine adrenal cortex and characterized. The detergent extract retained specific high-affinity binding sites for 125I-ANF. Scatchard analysis of the equilibrium binding data revealed a single class of binding site with a K-d of 1.8 nM and a maximum binding capacity of 2.5 pmol/mg of protein. The size of the 125I-ANF X receptor complexes was estimated to be 140,000 daltons by gel filtration on TSK gel G3000SW. Affinity labeling followed by electrophoresis under nonreducing conditions and autoradiography also revealed a single band of a similar size (Mr = 130,000); this band, however, migrated as a Mr = 70,000 species under reducing electrophoretic conditions. These results indicate that the ANF receptor, having a Mr of 130,000 - 140,000, is composed of disulfide-linked subunits and the ANF-binding site is located on the 70-kDa component.     

Solubilization and hydrodynamic properties of pig atrial muscarinic acetylcholine receptor in dodecyl beta-D-maltoside.

The pig atrial muscarinic acetylcholine receptor (mAcChR) has been solubilized from the membrane-bound state in high yield and in stable conformation by the non-ionic detergent dodecyl beta-D-maltoside (DBM). The yield and selectivity for receptor solubilization is dependent on the detergent/protein ratio during extraction. Extraction at 2 mg of DBM/mg of protein gave a 75% yield of solubilized receptor with a 1.5-fold enrichment. A double-extraction procedure, in which non-receptor protein was first extracted at 0.4 mg of DBM/mg of protein and mAcChR was selectively solubilized by a second extraction at 0.35 mg of DBM/mg of protein, gave a 50% overall yield and a 2.8-fold enrichment. Both preparations had a half-life of about 20 days on ice without addition of muscarinic ligands. Receptor stability was decreased by the presence of cations, particularly bivalent cations, and enhanced by the agonist carbachol. Dissociation constants for the interaction of the DBM-solubilized receptor with the antagonist L-quinuclidinyl benzilate (Kd = 223 pM) and the agonist carbachol (Kd = 100 microM) were similar to those for the digitonin/cholate-solubilized receptor. Pig atrial mAcChR purified in digitonin/cholate and exchanged into DBM displayed reliable hydrodynamic behaviour during sucrose density sedimentation in gradients of 2H2O and H2O and during gel filtration in Sephacryl S-300. DBM is thus the first detergent which will solubilize a stable form of the ligand-free mAcChR in yields similar to those with digitonin, and is the only stabilizing detergent thus far suitable for hydrodynamic studies. DBM is also likely to be similarly useful in studying other membrane proteins for which digitonin has been the solubilizing detergent of choice.     

Phospholipid requirement and pH optimum for the in vitro enzymatic activity of the E. coli P-type ATPase ZntA

Detergent solubilization and purification of the E. coli heavy metal P-type ATPase ZntA yields an enzyme with reduced hydrolytic activity in vitro. Here, it is shown that the in vitro hydrolytic activity of detergent solubilized ZntA is increased in the presence of negatively charged phospholipids and at slightly acidic pH. The protein-lipid interaction of ZntA was characterized by enzyme-coupled ATPase assays and fluorescence spectroscopy. Among the most abundant naturally occurring phospholipids, only phosphatidyl-glycerol lipids (PG) enhance the in vitro enzymatic ATPase activity of ZntA. Re-lipidation of detergent purified ZntA with 1,2-dioleoylphosphatidyl-glycerol (DOPG) increases the ATPase activity four-fold compared to the purified state. All other E. coli phospholipids fail to activate the ATPase. Among the phosphatidyl-glycerol family, highest activity was observed for 1,2-dioleoyl-PG followed by 1,2-dimyristoyl-PG, 1,2-dipalmitoyl-PG and 1,2-distearoyl-PG. Increasing intrinsic Trp fluorescence quantum yield upon relipidation of ZntA was used to determine a pH maximum for lipid binding at pH 6.7. The pH dependence of the lipid binding was confirmed by pH-dependent ATPase assays showing maximum activity at pH 6.7. The biophysical characterization of detergent solubilized membrane proteins crucially relies on the conformational stability and functional integrity of the protein under investigation. The present study describes how the E. coli ZntA P-type ATPase can be stabilized and functionally activated in a detergent solubilized system.     

Heterogeneity of Rat Brain Acetylcholinesterase: A Study by Gel Filtration and Gradient Centrifugation

Abstract: According to their solubilization properties, two classes of acetyl-cholinesterases (AChE) can be detected in the adult rat brain: a "soluble" species (easily solubilized without detergent), and a membrane-bound species (solubilized only in the presence of detergent). The latter was found to be homogeneous by gel filtration (Stokes radius 8.05 ± 0.35 nm) and sucrose gradient centrifugation (9.75 ± 0.2 S) in the presence of Triton X-100. The "soluble" AChE gives three stable species in the presence of the same detergent with Stokes radii and sedimentation constants of 10.9 ± 0.5 nm and 16 ± 2 S; 6.75 ± 0.30 nm and 10.7 ± 0.4 S; 5.37 ± 0.35 nm and 4.37 ± 0.1 S. Co-chromatography and co-sedimentation or the reduction and alkylation of disulfide bridges show that all the soluble species are different from the membrane-bound AChE. The possibility that soluble and membrane-bound AChE are completely different molecules is discussed.