# Binding of 125I-fasciculin to rat brain acetylcholinesterase. The complex still binds diisopropyl fluorophosphate

Pascale Marchot, Akila Khelif, Yong-Hua Ji, Pascal Mansuelle, Pierre E. Bougis

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Pascale Marchot, Akila Khelif, Yong-Hua Ji, Pascal Mansuelle, Pierre E. Bougis. Binding of 125I-fasciculin to rat brain acetylcholinesterase. The complex still binds diisopropyl fluorophosphate. Journal of Biological Chemistry, 1993, 268 (17), pp.12458-12467. hal-03261921

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# Binding of ¹²⁵I-Fasciculin to Rat Brain Acetylcholinesterase

THE COMPLEX STILL BINDS DIISOPROPYL FLUOROPHOSPHATE*

(Received for publication, October 20, 1992, and in revised form, February 22, 1993)

Pascale Marchot‡, Akila Khelif, Yong-Hua Ji§, Pascal Mansuelle, and Pierre E. Bougis

From the Centre National de la Recherche Scientifique, Unité de Recherche Associée URA 1455, Laboratoire de Biochimie, Faculté de Médecine, Secteur Nord, Université d'Aix-Marseille II, Bd. Pierre Dramard, 13916 Marseille Cedex 20, France

Iodination of fasciculin 3 (FAS3) from *Dendroaspis viridis* venom provided us with a fully active specific probe of fasciculin binding sites on rat brain acetylcholinesterase (AChE). Binding and inhibition are concomitant, as association and inhibition rate constants *k*₁ and *k*₂ are identical. The ¹²⁵I-FAS3·AChE complex dissociates very slowly (*t*₁/₂ = 48 h) and is characterized by a dissociation constant, *K*₉, of 0.4 pM. All the specific binding of ¹²⁵I-FAS3 to AChE is prevented by FAS3 as well as by the two other fasciculins, FAS1 and FAS2, from *D. angusticeps* venom (*K*₉ = 0.4, 14, and 25 pM, respectively). It is also prevented by propidium iodide, BW284C51, and d-tubocurarine, which bind to peripheral anionic sites of AChE, by Ca²⁺ and Mg²⁺, known to enhance AChE activity through an allosteric phenomenon and by acetylthiocholine concentrations which lead to excess substrate inhibition of the enzyme. Diisopropyl fluorophosphate and paraoxon, which inhibit AChE by phosphorylating the catalytic serine, have no effect on either the binding rate or the number of binding sites of ¹²⁵I-FAS3. O-Ethyl-S²-diisopropylaminoethyl methylphosphonothionate, however, which binds irreversibly to the AChE catalytic site but reversibly to a peripheral site, induces a 130% increase in the binding rate of ¹²⁵I-FAS3, without changing the total number of ¹²⁵I-FAS3 binding sites. Our results demonstrate that fasciculins bind on a peripheral site of AChE, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors and the substrate in excess bind. Since phosphorylation of the catalytic serine (esteratic subsite) by [1,3-³H]diisopropyl fluorophosphate can still occur on the FAS3·AChE complex, the structural modification induced by fasciculins may affect the anionic subsite of AChE catalytic site.

## Acetylcholinesterase (AChE)¹ of nerve and muscle tissues

* This work was supported in part by Direction Générale des Recherches et Etudes Techniques Contract 88/139 and by the Association Française contre les Myopathies. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed. Fax: 33-91-65-75-95.

§ Permanent address: Shanghai Inst. of Physiology, Chinese Academy of Sciences, 320 Yue Yang Rd., Shanghai, China 200031.

¹ The abbreviations used are: ACh, acetylcholine; AChE, acetylcholinesterase (EC 3.1.1.7); ATCh, acetylthiocholine; BSA, bovine serum albumin; BW284C51, 1,5-bis(4-allyldimethylammonium-phenyl)pentan-3-1-dibromide; DFP, diisopropyl fluorophosphate; DTNB, dithiobis-2-nitrobenzoic acid; HPLC, high-performance liquid chromatography; MPT, O-ethyl-S-[2-(diisopropylamino)ethyl] methylphosphonothionate; paraoxon, diethyl p-nitrophenyl phosphate.

is a polymorphic glycoprotein whose catalytic properties have been extensively studied (for a review see Rosenberry, 1975; Massoulié and Bon, 1982; Quinn, 1987). The crystal structure of *Torpedo californica* AChE, which has recently been solved (Sussman *et al.*, 1991), provides new evidence to explain the high catalytic efficiency of AChE, as well as the existence of multiple AChE inhibitors with multiple inhibition pathways. The AChE catalytic site was already known to be composed of an esteratic and an anionic subsite (or acetyl- and choline-binding sites, respectively). The site is now revealed to lie at the bottom of a deep and narrow gorge, which is lined by a high number of aromatic residues probably involved in a guidance mechanism facilitating two-dimensional diffusion of ACh from the gorge entrance to the buried active site (Sussman *et al.*, 1991). Additional binding sites for cationic ligands, remote from the active center, have been evidenced in biochemical studies and proposed to act as allosteric sites (Changéux, 1966; Taylor and Lappi, 1975; Epstein *et al.*, 1979). These peripheral anionic sites, which are now thought to be located at the rim area surrounding the gorge, would induce their long-range effects on active center reactivity through a relay mechanism along the aromatic surface of the gorge (Shafferman *et al.*, 1992).

A large number of organic compounds reversibly or irreversibly inhibit AChE (Long, 1963), which bind either to the esteratic or the anionic subsite of AChE catalytic site or to the peripheral site of the enzyme. Most of them are synthetic substances, sometimes bearing insecticidal properties (Murphy *et al.*, 1984). Few natural inhibitors of AChE are known and, to date, fasciculins are the only known proteinic AChE inhibitors. They are components of mamba snake venoms and have been shown to display a powerful inhibitory activity toward mammalian AChE. Up to now, three iso-fasciculins have been characterized: fasciculin 1 (FAS1) and fasciculin 2 (FAS2) from the venom of *Dendroaspis angusticeps* (green mamba) (Rodriguez-Ithurralde *et al.*, 1983), and toxin C from the venom of *Dendroaspis polylepis* (black mamba) (Joubert and Taljaard, 1978). The pharmacological and biochemical activities of FAS2 have been studied both *in vivo* and *in vitro* on various AChE-containing tissues (Karlsson *et al.*, 1984; Lin *et al.*, 1987). FAS2 inhibits several (but not all) AChEs from different sources with *K*ᵢ values of about 0.1 nM and has been shown to displace propidium, which is known to bind specifically to a peripheral anionic site of AChE (for a review see Harvey *et al.*, 1984; Cervenansky *et al.* 1991a).

In this study, we isolated from a *Dendroaspis viridis* venom a fasciculin (FAS3) which was found identical to toxin C from *D. polylepis* venom. To further define the relationships between binding site occupancy by fasciculins and rat brain AChE inhibition, we iodinated FAS3 and examined the ¹²⁵I-FAS3 binding properties. With respect to other AChE inhibitors, ¹²⁵I-FAS3 proved to be a new highly specific probe of
AChE peripheral site. As AChE inhibitors could be used as therapeutic agents against some cholinergic deficiencies, the detailed knowledge of the molecular mechanism of action of proteinic and peripheral-specific AChE ligands such as fasciculins is of particular interest.

## MATERIALS AND METHODS

**Venom and Reagents**—Crystallized *D. viridis* venom, batch 1963 no. 3, was from the Institut Pasteur (Paris, France). Sephadex G50 superfine was from Pharmacia (Uppsala, Sweden), and CM52 was from Whatman Biosystems (Maidstone (Kent), United Kingdom). The following items: HPLC-grade formic acid, TLC plastic sheets of precoated silica gel 60, analytical-grade chemicals and reagents were from Merck (Darmstadt, Federal Republic of Germany). Micropolyacrylamide A1700 TLC foils were from Schleicher and Schuell (Dassel, Federal Republic of Germany). Fluorescamine was from Hoffmann Laroche (Basel, Switzerland). HPLC-grade ammonium acetate was from Baker (Deventer, Holland), and HPLC-grade acetonitrile was from Touzard et Matignon (Vitry-sur-Seine, France). Enzymes were from the following sources: endoproteinase Arg-C, Boehringer (Mannheim, Federal Republic of Germany); *Staphylococcus aureus* V₈, Miles Laboratories (Elkhart, IN). Lactoperoxidase, carboxypeptidase A, BSA fraction IV, DTNB, propidium iodide, paraoxon, BW284C51, d-tubocurarine, and DFP were from Sigma. ATCh was from Serva (Heideberg, Federal Republic of Germany), and CaCl₂ and MgCl₂ were from Merck. MPT was a gift from Dr. J. Massoulié (Ecole Normale Supérieure, Paris, France). ¹²⁵I (2100 Ci mmol⁻¹) was from Amersham Corp. (Amersham, United Kingdom) and [1,3-³H]DFP (10 Ci mmol⁻¹) from New England Nuclear (Du Pont de Nemours, Les Ulis, France). Scintillation solution Pico-Fluor™15 was from Packard (Downers Grove, IL). Water was issued from a Millipore MilliRO/MilliQ system (Millipore, Bedford, MA).

**Purification of Fasciculins**—FAS1 and FAS2 from *D. angusticeps* venom were purified in the laboratory as described previously (Le Du *et al.*, 1989). FAS3 was purified as follows.

*D. viridis* venom, 5.3 g in 60 ml of water, was first dialyzed against water with spectrophore 3 (Spectrum Industries, Los Angeles, CA) for 4 h at 4 °C. After centrifugation at 10,000 × g for 35 min at 4 °C, the supernatant was made 0.1 M ammonium acetate, pH 8.5, and immediately gel-filtered through a set of four (5 × 100 cm) columns of Sephadex G50. Elution was performed with 0.1 M ammonium acetate, pH 8.5, at a flow rate of 60 ml h⁻¹. The fraction displaying the strongest AChE inhibitory activity was made 0.05 M ammonium acetate, pH 7.2, and loaded on a CM52 (2.5 × 45 cm) column equilibrated in the same solvent. Elution was performed with ammonium acetate, pH 7.2, as follows: 0.05 M for 22 h, a linear gradient from 0.05 to 0.5 M in 18 h, 0.5 M for 5 h, a linear gradient from 0.5 to 2 M in 5 h, and a 2 M final isocratic step. The flow rate was 60 ml h⁻¹. An additional chromatography of the AChE inhibitory fraction was carried out on CM52 in 0.2 M ammonium acetate, pH 7.2, at a flow rate of 20 ml h⁻¹. Finally, two HPLC procedures were successfully performed on a Waters system (Millipore): a reverse-phase HPLC on a semipreparative (10 × 250 mm) column prepacked with 5-µm ultrasphere-octyl (Beckman) with a linear gradient from 8 to 45% of acetonitrile in 0.15 M ammonium formate, pH 2.7, in 82 min at a flow rate of 5 ml min⁻¹ and an ion-exchange HPLC on a (6 × 150 mm) column prepacked with IEX-535-K carboxyspherogel-TSK (Beckman) in isocratic conditions with 0.3 M ammonium acetate, pH 5.0, containing 15% (v/v) acetonitrile, at a flow rate of 1 ml min⁻¹. A microtitration assay was set up to monitor the AChE inhibitory activity of the fractions obtained through the successive chromatographic steps. Each well of a Limbro 96 flat bottom well plate (Flow Laboratories, McLean, VA) contained, to a final volume of 150 µl, 50 mM NaH₂PO₄/Na₂HPO₄, pH 8.0, 0.1 mg ml⁻¹ BSA, 320 µg ml⁻¹ P₂, 0.6 mM DTNB, 0.3 mM ATCh (2 × Kₘ) and the sample. After stirring the microtitration plate for 15 min at 37 °C, absorbance at 414 nm was determined using a Titertek Multiscan MC spectrophotometer (Flow Laboratories).

**Protein Analysis**—Protein samples were hydrolyzed under vacuum for 20 or 70 h in the presence of 6 N HCl containing 1% (w/w) phenol, with a Waters Pico-Tag work station (Millipore). Amino acid analyses were carried out on a Beckman 6300 auto analyzer. Reduction and S-carboxymethylation of FAS3 (RCM-FAS3) were performed as described previously (Kopeyan *et al.*, 1975). CNBr cleavage of RCM-FAS3 at Met bonds was performed according to Gross (1967) by using 70% formic acid and adding solid CNBr with a molar excess of 70

over Met. Incubation was performed at room temperature in the dark for 24 h. Endoproteinase Arg-C (2% w/w) cleavage of RCM-FAS3 was performed in 0.05 M N-ethylmorpholine, pH 8.0, at 37 °C, for 5.5 h. S. aureus V₈ (10%, w/w) cleavage of RCM-FAS3 was performed in ammonium acetate, pH 4.0, at 37 °C for 24 h (Houmard and Drapeau, 1972). Peptides issued from both chemical and enzymatic cleavages were separated by TLC on silica gel 60 plastic sheets. For both CNBr and endoproteinase Arg-C peptides, the first dimension chromatography was performed by using an ammonia 25%/ethanol (40/60, v/v) solvent and the second dimension chromatography by using an ethanol/water/acetic acid (51/40/9, v/v) solvent. Only the first dimension chromatography was performed for V₈-peptides. After TLC, fluorescamine staining of peptides was performed according to Fishbein *et al.* (1980) and visualized at 366 nm. Peptides were extracted from the gel by 50% acetic acid or by the first dimension solvent. The extract was centrifuged at 10,000 × g for 10 min, filtered on glass wool then on 0.45 µm HV Millipore filters, and dried before being submitted to amino acid analysis and sequencing. Edman automated sequencing was performed on a Beckman system 890 M sequencer as described (Gatineau *et al.*, 1990; Bechis *et al.*, 1984). Manual dimethylaminoazobenzene-4'-isothiocyanate/phenylisothiocyanate method (Chang, 1983) was also used to determine peptide N-terminal sequence. The C-terminal sequence was obtained by hydrolysis of endoproteinase Arg-C-peptide₂₈⁻₆₁ with carboxypeptidase A (10%, w/w) in 0.1 M N-ethylmorpholine, pH 8.0, at 37 °C. Aliquots were removed at defined intervals, dried, and submitted to amino acid analysis.

**Iodination of FAS3**—FAS3 (5 µg) was enzymatically iodinated in the presence of 1 mCi of Na¹²⁵I with lactoperoxidase, then iodine in excess was removed by adsorption on Dowex Ac 1X10 (Bio-Rad) as already described (Martin *et al.*, 1987). A Crystal II Multidetector RIA system (Packard) was used for γ-radioactivity quantification. A specific activity of at least 1500 Ci mmol⁻¹ was routinely achieved. ¹²⁵I-FAS3 was stored 1 µM in 50 mM NaH₂PO₄/Na₂HPO₄, pH 7.5, 1 mg ml⁻¹ BSA, at 4 °C and used within 3 weeks.

The homogeneity of ¹²⁵I-FAS3 was attested by HPLC on a reverse-phase ultrasphere-octyl (4.5 × 250 mm) column (Beckman) by using a linear gradient from 10 to 50% of acetonitrile in 0.15 M ammonium formate, pH 2.7, in 40 min, at a flow rate of 1.5 ml min⁻¹ (Bougis *et al.*, 1986). The AChE inhibitory activity of the HPLC fractions was determined by using the microtitration assay above described. After multi-enzymatic digestion of ¹²⁵I-FAS3 (Marchot *et al.*, 1988), the respective proportions of ¹²⁵I-Tyr and ¹²⁵I-I₂-Tyr were further determined by HPLC on the same column by using a linear gradient from 5 to 25% of acetonitrile in 0.15 M ammonium formate, pH 2.7, in 20 min and at a flow rate of 1.5 ml min⁻¹. Carboxypeptidase A hydrolysis of ¹²⁵I-FAS3 was performed in 0.2 M N-ethylmorpholine, pH 8.5, at 37 °C, with an enzyme to ¹²⁵I-fasciculin ratio of 20 (w/w). Aliquots of 100 µl (2 × 10⁵ cpm) were taken at defined intervals, immediately mixed with 100 µl of 0.15 M ammonium formate, pH 2.7, containing 100 nmol of each Tyr, I-Tyr, and I₂-Tyr standards, then loaded on the reverse-phase ultrasphere-octyl (4.5 × 250 mm) column. Elution was performed with two successive linear gradients, from 5 to 25% and from 25 to 75% of acetonitrile in 0.15 M ammonium formate, pH 2.7, in 20 min and 25 min, respectively, at a flow rate of 1.5 ml min⁻¹. Fractions were collected and counted for radioactivity.

**Rat Brain AChE**—The crude synaptosomal fraction (P₂) of Wistar rat brain was prepared as described by Gray and Whittaker (1962). The protein content was determined using the Bio-Rad protein assay with BSA as standard. The specific AChE activity was 12.5 ± 3.0 nmol of ATCh h⁻¹ mg⁻¹ P₂ proteins (slightly depending on the preparation in use) and was stable for at least 80 h at 25 °C. Until use, P₂ was stored in liquid nitrogen without loss of AChE activity.

**AChE Activity Assays**—The AChE activity was determined at 25 °C by following the method of Ellman *et al.* (1961) with 1.5 mM ATCh (10 × Kₘ) and 0.32 mM DTNB in 100 mM NaH₂PO₄/Na₂HPO₄, pH 8.0, 0.1 mg ml⁻¹ BSA (buffer I) to a final volume of 1.5 ml. Two successive determinations of ΔA min⁻¹ (interval time, 40 s) were performed with a Unicam 8700 spectrophotometer (Cambridge, United Kingdom) equipped with the optional kinetic program. Data points correspond to duplicates that differed by less than 10%. For inhibition kinetic assays, FAS3 was incubated with P₂ (0.1 mg ml⁻¹) in buffer I. At defined intervals, two aliquots of 250 µl were taken and immediately assayed for residual AChE activity.

**¹²⁵I-FAS3 Binding Kinetic Assays**—Binding assays were performed at 25 °C in rhesus tubes containing 10 mM Tris-HCl, pH 7.5, 100 mM NaCl, 0.1 mg ml⁻¹ BSA (buffer II), by adding successively ¹²⁵I-FAS3 and P₂, to a final volume of 250 µl. For nonspecific binding determi-
**Chemical Characterization of FAS3**—The first 25 residues (except residues 5, 7–10, and 20–22), and residue in position 32, were determined on RCM-FAS3 (Fig. 2a). The last 3 residues were obtained by the action of carboxypeptidase A on endoproteinase Arg-C-peptide₂₈⁻₆₁ (Fig. 2g). The CNBr-, endoproteinase Arg-C- and V₈-peptides were purified by TLC. Amino acid analysis and/or sequencing of these peptides yielded the necessary overlaps and allowed the complete amino acid sequence of FAS3 to be obtained (Fig. 2, b–h).

**Characterization of ¹²⁵I-FAS3**—With reference to the specific radioactivity obtained (1500 Ci mmol⁻¹), the iodine incorporation was 0.7 atom of iodine/molecule of FAS3. ¹²⁵I-FAS3 eluted from reverse-phase HPLC as a single peak with a 5-min delay with regard to the retention time of FAS3; AChE inhibitory activity co-eluted with the radioactivity. After multi-enzymatic digestion of ¹²⁵I-FAS3 and subsequent analysis using reverse-phase HPLC (Fig. 3A), at least 95% of the radioactivity was recovered as ¹²⁵I-Tyr (data not shown).

FAS3 has 4 Tyr residues at positions 4, 23, 47, and C-terminal 61 (Fig. 2). Thus, the location of ¹²⁵I-Tyr was investigated by reverse-phase HPLC after carboxypeptidase A hydrolysis of ¹²⁵I-FAS3. Fig. 3B shows the kinetics of the radioactivity released as ¹²⁵I-Tyr. After 60 min of hydrolysis, 50% of the radioactivity of the sample submitted to chromatography was released as ¹²⁵I-Tyr, whereas the remaining radioactivity still eluted as ¹²⁵I-FAS3. Therefore, one-half of the radioactivity was incorporated on Tyr⁶¹. When compared with FAS3, FAS2 has only 3 Tyr residues at positions 4, 23, and C-terminal 61 (Tyr⁴⁷ is replaced by Asn⁴⁷ in FAS2). Then, FAS2 was iodinated in the same experimental conditions as FAS3 (specific radioactivity achieved: 1500 Ci mmol⁻¹). When ¹²⁵I-FAS2 was submitted to carboxypeptidase A analysis, 100% of the radioactivity of the sample was recovered as ¹²⁵I-Tyr after as little as 30 min of incubation. FAS2 was thus iodinated on Tyr⁶¹ only, Tyr⁴ and Tyr²³ being not reactive. As both Tyr⁴ and Tyr²³ in FAS3 were likely not to be reactive either, the half of the radioactivity remaining after carboxypeptidase A hydrolysis of ¹²⁵I-FAS3 should be incorporated on Tyr⁴⁷, i.e. the only additional residue which may be iodinated. Two hypotheses arise from these results: (i) one-half of FAS3 was iodinated on both Tyr⁴⁷ and Tyr⁶¹, the second half being not iodinated; ii) one-half of FAS3 was iodinated on Tyr⁴⁷ and the second half on Tyr⁶¹. Since the AChE inhibitory activity co-eluted with the radioactivity only, the

first hypothesis was not valid. Finally, to compare the reactivity of Tyr⁴⁷ and Tyr⁶¹, FAS3 was iodinated in the presence of 10 times less Na¹²⁵I (specific radioactivity achieved: 140 Ci mmol⁻¹). Again, carboxypeptidase A hydrolysis revealed that 50% of the radioactivity was incorporated on Tyr⁶¹, thus suggesting that Tyr⁴⁷ and Tyr⁶¹ were equally reactive regardless of the ratio of ¹²⁵I/FAS3 used. It was not possible to differentiate ¹²⁵I-Tyr⁴⁷-FAS3 from ¹²⁵I-Tyr⁶¹-FAS3 with reverse-phase HPLC; the mixture was therefore used all through the study.

**Kinetic Experiments**—The rate of binding of ¹²⁵I-FAS3 to AChE was studied for concentrations ranging from 45 pM to 1 nM. Fig. 4 shows the results obtained for 45 pM (Fig. 4A) and 445 pM (Fig. 4B). A single association process was observed regardless of the ¹²⁵I-FAS3 concentration used. Plotting the data according to Equation 1 allowed the pseudo-first-order kinetic constant, $k_{obs/bind}$, to be determined. Plotting $k_{obs/bind}$ against free ¹²⁵I-FAS3 concentrations according to Equation 3a yielded the second-order kinetic constant of the association process, $k_{+1} = (9 \pm 1) \times 10^6 \text{ M}^{-1} \text{ s}^{-1}$ (Fig. 4C). Theoretically, the first-order kinetic constant $k_{-1}$ should be determined from the intercept on the y axis of the plot, according to Equation 3a. In the present case, however, the intercept was so close to zero ((1 ± 3) × 10⁻⁴ s⁻¹) that $k_{-1}$ could only be roughly estimated as <10⁻⁴ s⁻¹.

To verify if ¹²⁵I-FAS3 is as a fully active AChE ligand as FAS3, we studied the rate of inhibition of AChE activity by FAS3 for concentrations ranging from 0.1 to 1.5 nM. Fig. 5 shows the results obtained for 0.1 nM (Fig. 5A) and 1.25 nM (Fig. 5B). A single inhibition process was observed regardless of the FAS3 concentration used. About 10% of the initial AChE activity remained not inhibited, even for the highest FAS3 concentrations. Taking into account this value as an offset value, plotting the data according to Equation 2 allowed the pseudo-first-order kinetic constant, $k_{obs/inh}$, to be determined. Plotting $k_{obs/inh}$ against FAS3 concentrations according to Equation 3b yielded the second-order kinetic constant of the inhibition process $k_i = (10.9 \pm 0.7) \times 10^6 \text{ M}^{-1} \text{ s}^{-1}$ (Fig. 5C). In that case, $k_{-i}$ could only be roughly estimated as <10⁻⁴ s⁻¹ ((3 ± 6) × 10⁻⁴ s⁻¹).

The dissociation process of ¹²⁵I-FAS3 initially bound to AChE was visualized after the addition of a 1000-fold excess of FAS3. Plotting the data according to Equation 4 yielded the first-order kinetic constant $k_{-1} = (4 \pm 1) \times 10^{-6} \text{ s}^{-1}$, i.e. a

<table><caption>**FIG. 2.** Determination of the amino acid sequence of FAS3 and comparison with the known sequences of toxin C (Joubert and Taljaard, 1978), FAS2 (Viljoen and Botes, 1973), and FAS1 (Ducancel et al., 1991). *a*, Edman automated sequencing performed on RCM-FAS3; *b*, on CNBr-peptide₃₄⁻₆₁; *c*, on S. aureus V₈-peptide₆₀⁻₆₁; *d*, on endoproteinase Arg-C-peptide₁⁻₁₁; *e*, on endoproteinase Arg-C-peptide₁₂⁻₂₄; *f*, on endoproteinase Arg-C-peptide₂₈⁻₆₁; *g*, carboxypeptidase A hydrolysis of endoproteinase Arg-C-peptide₂₈⁻₆₁; *h*, sequence of endoproteinase Arg-C-peptide₂₅⁻₂₇ as determined from amino acid composition. \*, indicates differences between the sequences.</caption><tr><td>(a)</td><td>TICY.H....RAILKDCGE....YRK....K....................</td></tr><tr><td>(b)</td><td>....................VLGRGCGCPPGDDYLEVK...........</td></tr><tr><td>(c)</td><td>....................VKCCTSPDKCNY....................</td></tr><tr><td>(d)</td><td>TICYSHTTTS....................</td></tr><tr><td>(e)</td><td>AILKDCGEN.CY....................</td></tr><tr><td>(f)</td><td>RHPPKMVL....................</td></tr><tr><td>(g)</td><td>....................CNY....................</td></tr><tr><td>(h)</td><td>KSR....................</td></tr><tr><td>FAS3</td><td>TICYSHTTTSRAILKDCGENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKCNY</td></tr><tr><td>ToxC</td><td>TICYSHTTTSRAILKDCGENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKCNY</td></tr><tr><td>FAS2</td><td>TMCYSHTTTSRAILTNCGENSCYRKSRRHPPKMVLGRGCGCPPGDDNLEVKCCTSPDKCNY</td></tr><tr><td>FAS1</td><td>TMCYSHTTTSRAILTNCGENSCYRKSRRHPPKMVLGRGCGCPPGDDYLEVKCCTSPDKCNY</td></tr><tr><td></td><td>1 1 2 3 4 5 6 0</td></tr></table>
<figure><img src="image_2.png" /></figure>

FIG. 3. Characterization of the products obtained after carboxypeptidase A hydrolysis of ¹²⁵I-FAS3. A, reverse-phase HPLC elution diagram of a sample constituted of Tyr, I-Tyr, I₂-Tyr and, as located by radioactivity, ¹²⁵I-FAS3; B, kinetics of ¹²⁵I-Tyr release upon incubation of ¹²⁵I-FAS3 (●) (1500 Ci mmol⁻¹), ¹²⁵I-FAS3 (O) (140 Ci mmol⁻¹), and ¹²⁵I-FAS2 (■) (1500 Ci mmol⁻¹) with carboxypeptidase A. 100% of radioactivity (y axis) corresponds to the radioactivity of the sample submitted to chromatography (2 × 10⁵ cpm).

$t_{1/2}$ value of 48 h (Fig. 6). The true dissociation constant of the ¹²⁵I-FAS3·AChE complex was derived from the dissociation and association kinetic constants, i.e. $K_d = k_{-1}/k_1 = 0.4$ pM.

*Competition Binding Experiments*—The effect of fasciculins FAS1, FAS2, and FAS3, as well as of the anti-cholinesterase agents BW284C51, propidium iodide, DFP, paraoxon, *d*-tubocurarine, and MPT, on the binding of ¹²⁵I-FAS3 to AChE after their preincubation with $P_2$ was studied on the basis of the decrease in the initial binding rate of ¹²⁵I-FAS3 (Fig. 7). Whereas 1 mM of either DFP or paraoxon totally inhibits AChE, increasing concentrations of DFP or paraoxon, up to 1 mM, had no significant effect on the initial binding rate of ¹²⁵I-FAS3 (data not shown). On the contrary, preincubation of $P_2$ in the presence of increasing concentrations of FAS3, FAS2, and FAS1, as well as of BW284C51, propidium iodide, and *d*-tubocurarine, provoked a significant decrease in the initial binding rate of ¹²⁵I-FAS3. In each case, the inhibition of ¹²⁵I-FAS3 binding was complete and occurred within 2 orders of magnitude of the anti-cholinesterase agent concentration. The half-effect ($K_{0.5} = K_d$, see "Materials and Methods") was obtained for 0.45 ± 0.05 pM of FAS3, 14 ± 2 pM of FAS1, 25 ± 3 pM of FAS2 (each value being the average of three independent experiments) (Fig. 7A), 3.2 nM of BW284C51, 0.1 µM of *d*-tubocurarine, and 5.6 µM of propidium iodide (Fig. 7B).

The effect of $CaCl_2$ and $MgCl_2$, as well as of ATCh, on ¹²⁵I-FAS3 binding is also reported on Fig. 7B. The rate was found

<figure><img src="image_3.png" /></figure>

FIG. 4. Kinetics of formation of the ¹²⁵I-FAS3·AChE complex. A, association kinetics performed in the presence of 45 pM of ¹²⁵I-FAS3 and 0.1 mg ml⁻¹ $P_2$ proteins; the data were fitted to Equation 1, yielding $k_{obs/bind} = (7.2 \pm 0.3) \times 10^{-4}$ s⁻¹; inset, semilogarithmic plot of the data; B, association kinetics performed in the presence of 445 pM of ¹²⁵I-FAS3 and 0.1 mg ml⁻¹ $P_2$ proteins; the data were fitted to Equation 1, yielding $k_{obs/bind} = (4.8 \pm 0.3) \times 10^{-3}$ s⁻¹; inset, semilogarithmic plot of the data; C, $k_{obs/bind}$ values were plotted against the ¹²⁵I-FAS3 free concentrations according to Equation 3a, yielding the second-order rate constant $k_2 = (9 \pm 1.0) \times 10^6$ M⁻¹ s⁻¹.

to decrease within 2 orders of magnitude of the salt concentration, up to almost total inhibition of ¹²⁵I-FAS3 binding ($v/v_0 < 10\%$ for 100 mM). Half-effect was obtained for 12.6 mM of both $CaCl_2$ and $MgCl_2$. As well, a significant decrease in the binding rate was observed for ATCh concentrations higher than 1.0 mM, up to a 40% decrease for 10 mM ATCh. In an attempt to correlate this phenomenon to the effect of excess substrate AChE inhibition, the $P_2$ enzymatic activity was assayed for ATCh concentrations ranging from 1.5 µM to 15.5 mM. As expected, the AChE specific activity significantly decreased for ATCh concentrations higher than 1.0 mM, up to a 45% decrease for the highest concentration 15.5 mM (data not shown).

The effect of MPT on the initial binding rate of ¹²⁵I-FAS3 to AChE was also assayed (Fig. 7B, inset). The rate was found to increase up to 130% from 0.1 nM and within 2 orders of magnitude of the MPT concentration. For MPT concentrations higher than 10 nM, the rate drew nearer the 100% value but never joined it, even for the highest concentration tested (0.1 mM).

*Titration of AChE and Turnover Number Determination*—The number of ¹²⁵I-FAS3 binding sites on $P_2$ was studied for ¹²⁵I-FAS3 concentrations ranging from 25 pM to 1 nM (Fig. 8A). Nonspecific binding accounted for about 25% of total binding. The ¹²⁵I-FAS3 specific binding was saturable and the plateau of the titration curve was used to estimate the number
<figure><img src="image_4.png" /><figcaption>FIG. 5. Kinetics of AChE inhibition by FAS3. A, residual AChE activity of 250-µl aliquots of the incubation mixture containing 0.1 nM of FAS3 and 0.1 mg ml⁻¹ P₂ proteins; the data were fitted to Equation 2, yielding *k*<sub>obs/inh</sub> = (12.3 ± 0.8) × 10⁻⁴ s⁻¹; inset, semilogarithmic plot of the data; B, residual AChE activity of 250-µl aliquots of the incubation mixture containing 1.25 nM of FAS3 and 0.1 mg ml⁻¹ P₂ proteins; the data were fitted to Equation 2, yielding *k*<sub>obs/inh</sub> = (13 ± 2) × 10⁻³ s⁻¹; inset, semilogarithmic plot of the data; C, *k*<sub>obs/inh</sub> values were plotted against FAS3 initial concentrations according to Equation 3b, yielding the second-order rate constant *k*ᵢ = (10.9 ± 0.7) × 10⁶ M⁻¹ s⁻¹.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>FIG. 6. Kinetics of dissociation of the ¹²⁵I-FAS3·AChE complex. ¹²⁵I-FAS3 was 300 pM and was initially allowed to bind to 0.1 mg ml⁻¹ P₂ proteins. A 1000-fold excess of FAS3 was added to the mixture to initiate the complex dissociation and then aliquots of 250 µl were taken at defined intervals and the amount of bound ¹²⁵I-FAS3 determined. The data were fitted to Equation 4, yielding the first-order rate constant *k*<sub>-1</sub> = (4 ± 1) × 10⁻⁶ s⁻¹; inset, semilogarithmic plot of the data.</figcaption></figure>

of binding sites $B_{\max} = 0.42 \pm 0.01 \text{ pmol mg}^{-1}$. Taking into account the specific AChE activity of the preparation (15.5 µmol of ATCh h⁻¹ mg⁻¹), the turnover number per site was $3.7 \times 10^7$ mol of ATCh hydrolyzed h⁻¹. The total number of ¹²⁵I-FAS3 binding sites was found unchanged when deter-

mined in the presence of MPT in excess preincubated with $P_2$.

Titration of the number of [1,3-³H]DFP binding sites on $P_2$ is reported on Fig. 8B. Nonspecific binding accounted for about 10% of total binding. The specific binding was saturable with $B_{\max} = 0.52 \pm 0.01 \text{ pmol mg}^{-1}$, and the turnover number per site was calculated to be $3.0 \times 10^7$ mol of ATCh h⁻¹. The total number of [1,3-³H]DFP binding sites was not significantly changed when determined in the presence of FAS3 in excess preincubated with $P_2$. Moreover, the kinetic binding of [1,3-³H]DFP (4 nM) to AChE was also found unchanged when assayed in the presence of FAS3 (4 nM) preincubated with $P_2$ for 30 min (data not shown).

The variation of the residual AChE activity of $P_2$ was assayed as a function of either FAS3 or MPT concentration (Fig. 8C). After 4 h of incubation, the plot obtained for FAS3 was a straight line which, when extrapolated, yielded $B_{\max} = 0.4 \text{ pmol mg}^{-1}$ as the number of FAS3 binding sites and a turnover number per site of $3.6 \times 10^7$ mol of ATCh h⁻¹. An identical result was obtained after an overnight incubation. In contrast, the plot obtained for MPT largely diverged from linearity in the case of 4 h of incubation (data not shown). When incubation was extended to overnight, the plot diverged as early as at 50% inhibition. Extrapolation to zero activity based on initial 50% inhibition data points yielded the number of MPT binding sites $B_{\max} = 1.0 \text{ pmol mg}^{-1}$ and a turnover number per site of $1.55 \times 10^7$ mol of ATCh h⁻¹. This result was found unchanged after longer incubation.

DISCUSSION

When compared with the sequences of fasciculins FAS1 and FAS2 from *D. angusticeps* venom and toxin C from *D. polylepis* venom, the sequence of FAS3 is identical with that of toxin C (Fig. 2). The possibility for the presence of identical fasciculins in venoms of mambas of two different species has to be questioned. Moreover, it should be noted that the true origin of the venom that we used has been already questioned with regard to the isolation of α-neurotoxins (Bechis *et al.*, 1976). *D. viridis* venom has been reported to contain several anti-cholinesterase toxins (Harvey *et al.*, 1984). *A. viridis* venom of certified origin (Latoxan, Rosans, France), as well as several other individual *D. viridis* venoms, were also tested for AChE inhibitory activity. No such activity, however, was found in these venoms (Marchot, 1986). When venoms are collected, misidentification of snake species and subspecies should be considered as of outstanding importance.

Iodination of FAS3 with mild procedure provided us with a fully active specific probe of exceptionally high affinity for an AChE peripheral binding site. This site is, at least partly, shared with the respective binding sites of some other inhibitors, as well as of substrate in excess. Characterization of ¹²⁵I-FAS3 showed that, among the 4 Tyr residues of FAS3 (in positions 4, 23, 47, and 61), only Tyr⁴⁷ and Tyr⁶¹ may be iodinated by using lactoperoxidase catalysis (Fig. 3). For the identically shaped elapid toxins, cardiotoxins and short α-neurotoxins, it has been proved that Tyr²³, next to Cys²² (fasciculin numbering), cannot be enzymatically iodinated because of its buried location within the molecule (Sato and Tamiya, 1970; Rochat *et al.*, 1977; Bougis *et al.*, 1983). For similar reasons, Tyr²³ and Tyr⁴ (next to Cys³) of fasciculins were likely not to be accessible to iodination, as evidenced from recent resolution of the three-dimensional x-ray structure of FAS1 (Le Du *et al.*, 1992). Characterization of ¹²⁵I-FAS2, which we demonstrated to be iodinated only on Tyr⁶¹, as Tyr⁴⁷ is replaced by Asn⁴⁷, confirms this assumption.

Kinetic studies conducted with ¹²⁵I-FAS3 and FAS3 on
<figure><img src="image_6.png" /><figcaption>FIG. 7. Competition binding experiments between ¹²⁵I-FAS3 and either FAS3, FAS2, and FAS1, or various AChE effectors, or acetylthiocholine. A, the initial binding rate of 100 pM ¹²⁵I-FAS3 to 0.1 mg ml⁻¹ P₂ proteins was determined in the absence (v₀) and in the presence (v) of increasing concentrations of FAS3 (■), FAS1 (●), or FAS2 (▲), preincubated with P₂ during 30 min. B, the same experiment was performed with increasing concentrations of either BW284C51 (▼), d-tubocurarine (●), propidium iodide (♦), CaCl₂ (○), or MgCl₂ (Δ) preincubated with P₂ during 30 min or of ATCh (□) not preincubated; inset, the experiment was performed in the presence of increasing concentrations of MPT, preincubated with P₂ during 30 min.</figcaption></figure>

binding to AChE and on AChE inhibition, respectively, were both well fitted by single exponential processes (Figs. 4 and 5). The great similarity of the two second-order kinetic constants, $k_1$ and $k_i$, means that the rate of FAS3 association to AChE is not modified upon iodination and that binding and inhibition are concomitant processes in the time scale of the respective experiments. The kinetics of ¹²⁵I-FAS3 dissociation, the data of which were well fitted by a single exponential decay equation, clearly indicate an extremely slow process ($t_{1/2}$ = 48 h) (Fig. 6). This result is consistent with reports on the apparent irreversibility of the action of fasciculins on murine diaphragm preparations (Anderson *et al.*, 1985) and on red blood cell AChE (Lin *et al.*, 1987). The dissociation constant of the ¹²⁵I-FAS3·AChE complex, which was determined from the kinetic constants (Figs. 4 and 6), is exceptionally low ($K_d$ = 0.4 pM). As the same $K_d$ value was determined for the FAS3·AChE complex by quantifying, with ¹²⁵I-FAS3, the amount of binding sites not occupied by FAS3 (Fig. 7A), the affinity of FAS3 for AChE is not either changed upon iodination. The dissociation constants of complexes formed with FAS1 and FAS2 (14 and 25 pM, respectively) are significantly higher than that of the complex formed with FAS3 (Fig. 7A) and in agreement with the $K_i$ value of 16 pM kinetically determined for FAS1 on liposomal bovine brain AChE (Puu and Koch, 1990) and with the "apparent" $K_i$ value of 35 pM reported in an equilibrium study on rat brain AChE inhibition by FAS2 (Karlsson *et al.*, 1984). FAS1 and FAS2 differ by the single amino acid substitution Tyr/Asn at position 47, which may account for their 2-fold difference in affinity, the Tyr residue slightly enhancing the stability of the FAS·AChE complex. In contrast, FAS3 differs from both FAS1 and FAS2 by substitutions in positions 2 (Ile/Met), 15 (Lys/Thr) and 16 (Asp/Asn), respectively (Fig. 2). The second and the third substitutions, which bring not only local differences in charge, but also the long side chain of the Lys residue, are more likely to account for the 35-fold higher affinity of FAS3 as compared with FAS1. These results, together with the partial loss of activity occurring upon chemical modification of Arg and Lys residues of FAS2 (Cervenansky *et al.*, 1991b), support the hypothesis of multipoint attachment of fasciculins on AChE, according to which several residues of

the fasciculin molecule are involved simultaneously in the interaction with the target enzyme. Such a mode of attachment, which has already been described for the interaction of snake venom α-neurotoxins with their binding sites on nicotinic ACh receptor (Faure *et al.*, 1983; Martin *et al.*, 1983), most probably explains the exceptionally high affinity of FAS3 for rat brain AChE. Such an affinity is, up to now, the highest reported for fasciculins.

Incidentally, the fact that all our data were well fitted by single exponential processes (kinetic data) or by single sigmoidal processes (competition data) strongly support the presence of a single AChE form in our membrane preparation. In rat brain, indeed, membrane-bound AChE has been reported to be made exclusively of the globular G4 form (Rieger and Vigny, 1976; Bisso *et al.*, 1991). The four G4 subunits, the catalytic properties of which are essentially identical (Vigny *et al.*, 1978; Massoulié and Bon, 1982), thus appear to be also equally susceptible to fasciculins.

DFP and paraoxon are organophosphorus compounds which irreversibly inhibit AChE by phosphorylating the esteratic serine in the catalytic site. On the contrary, propidium iodide, d-tubocurarine, and BW284C51 are known to reversibly inhibit AChE by acting through AChE anionic sites. Propidium iodide selectively binds to a peripheral anionic site (Taylor and Lappi, 1975) which is remote from the AChE catalytic site and has a regulatory role (allosteric site). d-Tubocurarine also binds to such a site, sometimes with complex behavior depending on the ionic strength of the medium (Changeux, 1966; Mooser and Sigman, 1974; Zorko and Pavlic, 1986; Friboulet *et al.*, 1990). BW284C51 has been thought to bind selectively to the anionic subsite of the catalytic site (Austin and Berry, 1953); however, an additional peripheral interaction between AChE and BW284C51 has been reported (Friboulet *et al.*, 1986). Cations, and particularly bivalent ions, have been shown to induce an increase in substrate affinity and reaction velocity of AChE (Changeux, 1966; Robaire and Kato, 1974), but a decrease both in the inhibition due to some competitive inhibitors and in excess substrate inhibition of AChE; interaction with peripheral anionic centers was suggested. The competition binding experiments performed with the AChE inhibitors or effectors above cited allowed us to
<figure><img src="image_7.png" /><figcaption>FIG. 8. Titration of the binding sites present on P₂. The P₂ preparation (0.1 mg ml⁻¹, 15.5 µmol of ATCh h⁻¹ mg⁻¹) was incubated during 4 h in the presence of increasing concentrations of inhibitor; A, determination of the number of ¹²⁵I-FAS3 binding sites: total (O), nonspecific (□), and specific (●) binding; Δ represents the total binding of ¹²⁵I-FAS3 in the presence of MPT (10⁻⁷ M) preincubated with P₂ for 2 h; B, determination of the number of [1,3-³H]DFP binding sites: total (O), nonspecific (□), and specific (●) binding; Δ represents the total binding of [1,3-³H]DFP in the presence of FAS3 (10⁻⁷ M) preincubated with P₂ for 2 h; C, residual AChE activity of P₂ as a function of either FAS3 (●) or MPT (O) concentration. Dashed line shows the extrapolation to zero activity based on initial 50% inhibition data points. The double scale (x axis) permits comparison with the y axis of the data plots reported in A and B.</figcaption></figure>

examine the relationships between binding site occupancy by ¹²⁵I-FAS3 and AChE inhibition. Although both DFP and paraoxon fail to prevent ¹²⁵I-FAS3 from binding to its site, inhibitors acting through AChE peripheral anionic sites, i.e. BW284C51, *d*-tubocurarine, and propidium iodide, are able to compete with ¹²⁵I-FAS3 (Fig. 7B). The *K*<sub>d</sub> values which we obtained are in agreement with previously reported *K*<sub>d</sub> or *K*<sub>i</sub> values: for BW284C51, 3.2 nM has to be compared with 1.2 nM reported as a maximal *K*<sub>i</sub> value (Austin and Berry, 1953); for *d*-tubocurarine, 0.1 µM has to be compared with 3.0 µM as determined by back-titration of propidium iodide fluorescence in the case of *T. californica* AChE (Taylor and Lappi, 1975) but in low ionic strength conditions in which *d*-tubocurarine is also reported to bind at the ionic subsite of the AChE catalytic site (Mooser and Sigman, 1974); for propidium iodide, 5.6 µM has to be compared with 3.0 µM, as determined in identical ionic strength conditions (Berman and Taylor, 1978), with 1.1 µM, as determined in low ionic strength conditions (Berman and Leonard, 1990), or with 0.3 µM (Taylor and Lappi, 1975) or 0.1 µM (Berman *et al.*, 1981). The extinction, by FAS2, of the fluorescence of propidium iodide

bound to AChE has been reported (Karlsson *et al.*, 1984). However, extinction was not complete (about 20% of the signal remained) until Ca²⁺ was added, indicating mutually exclusive binding sites for Ca²⁺ and propidium. Fasciculins are also likely to share, at least partly, such sites, since Ca²⁺ and Mg²⁺ prevent more than 90% of ¹²⁵I-FAS3 binding on rat brain AChE (Fig. 7B). Similar results were reported for inhibition of *Bungarus multicinctus* venom AChE by FAS2 and toxin C (Lin *et al.*, 1987); Mg²⁺ was also reported to displace human erythrocyte AChE from a FAS2-cyanogen bromide-activated Sepharose 4B column (Sindhuphak *et al.*, 1988). Those results confirm that fasciculins bind on a peripheral site of the enzyme, distinct from the catalytic site and, at least partly, common with the sites on which some cationic inhibitors bind.

Among cholinesterases, only AChE presents an inhibition by excess of substrate with ACh as well as ATCh, for concentrations above 2 mM (Augustinsson, 1963; Cohen and Oosterbaan, 1963). Rat brain AChE displays such a bell-shaped activity curve, the AChE activity starting to decrease significantly for ATCh concentrations higher than 1.6 mM (data not shown). An inhibition of the initial rate of ¹²⁵I-FAS3 binding to AChE was observed for ATCh concentrations higher than 1.5 mM (Fig. 7B). These results, which are in favor of a direct relation between the two phenomena, strongly support early proposals (Changeux, 1966; Aldridge and Reiner, 1969) as well as recent findings (Radic *et al.*, 1991) according to which a peripheral anionic center serves as supplementary binding site for ACh. Moreover, since the half-effect of the competition process was nearly obtained around 10–15 mM ATCh, the decreased ¹²⁵I-FAS3 binding rate that we observed could not be generated by the product inhibitor thiocholine, the concentration of which reaches a maximal value of 0.2 mM in our experimental conditions. Therefore, the peripheral anionic site on which ¹²⁵I-FAS3 binds would also be partly common with the additional substrate binding site responsible for the excess substrate inhibition of AChE. The AChE peripheral site, as it is usually referred to in the literature, is likely to consist of a matrix of partially overlapping loci corresponding to the diverse known peripheral ligands. For one of these loci to be entirely shared by fasciculins and peripheral ligands would be of interest, especially since fasciculins, because of their size, may be expected to cover a larger area of the AChE surface than low molecular weight ligands.

The number of ¹²⁵I-FAS3 binding sites on P₂ (0.42 pmol mg⁻¹) yielded a turnover number of the membrane preparation of 3.7 × 10⁷ mol of ATCh h⁻¹ (Fig. 8A). Both values were confirmed by titrating P₂ with FAS3 (Fig. 8C). However, the number of [1,3-³H]DFP binding sites on P₂ was found to be 20% higher (0.52 pmol mg⁻¹) (Fig. 8B). As DFP is known as an active serine inhibitor (Neurath, 1984), the additional DFP binding sites are likely to belong to some serine hydrolases other than AChE. Incidentally, such hydrolases may be responsible for the 10% ATCh hydrolysis that remains insensitive to high FAS3 concentrations (Fig. 5). Titration of the number of MPT binding sites (1.00 pmol mg⁻¹), however, led to a turnover number of 1.55 × 10⁷ mol of ATCh h⁻¹ (Fig. 8C). Although very close to that of 1.32 × 10⁷ mol of ATCh h⁻¹ reported by Vigny *et al.* (1978) for rat brain AChE, this value is 2-fold lower than that determined with [1,3-³H]DFP, ¹²⁵I-FAS3, or FAS3. MPT has been reported to interact not only with the catalytic site of AChE, as DFP does (Vigny *et al.*, 1978), but also with a second site, which may overlap or be linked to the peripheral anionic site of AChE, since it shares common features with the BW284C51 and propidium binding
sites (Friboulet *et al.*, 1986, 1990). The binding capacity which we obtained upon titration of AChE with MPT may thus account for both MPT binding sites, leading to a 2-fold underestimation of the turnover number. Furthermore, a decrease in the MPT effect at the active site has been reported to occur upon additional binding of MPT on its peripheral site. The curved shape of our MPT titration plot may therefore proceed from such a phenomenon, i.e. an increase in the MPT concentration needed for a given AChE inhibition, as occupancy of the peripheral site lowers the inhibition effect due to catalytic site occupancy. As already postulated (Friboulet *et al.*, 1990), this phenomenon may be explained by conformational changes in the AChE structure. In contrast to MPT, and more specifically than DFP, fasciculins form a 1:1 complex with the AChE catalytic monomer and, thus, prove to be more appropriate than organophosphorus compounds for specific titration of AChE.

In contrast with the other inhibitors and effectors above cited, MPT induces an increase in the ¹²⁵I-FAS3 binding rate (inset, Fig. 7B). Since the total number of ¹²⁵I-FAS3 binding sites was found unchanged after preincubation of P₂ with MPT (Fig. 8A), this increase must proceed from an increase in the value of second-order kinetic constant k₁ for ¹²⁵I-FAS3. We can postulate that the accelerated binding of ¹²⁵I-FAS3, in the presence of MPT, is due to a local change in the structure of the FAS3 binding site. This change may concern at least some AChE residues involved in the approach and docking of fasciculins. The fact that high concentrations in MPT cancel the accelerated binding phenomenon, but fail to prevent ¹²⁵I-FAS3 from binding to its site, argues for allosteric changes rather than for overlapping binding sites.

Recent resolution of the x-ray structure of *Torpedo* AChE (Sussman *et al.*, 1991) evidenced a deep and narrow gorge, the bottom of which contains the catalytic triad (Ser²⁰⁰, His⁴⁴⁰ Glu³²⁷). The gorge is lined with a high number of aromatic residues which may provide a "guidance mechanism" for ACh molecules. These residues, which make the ACh binding site hydrophobic rather than anionic, may also serve as a relay of molecular events from the peripheral binding sites, presumably located at the rim of the gorge, to the active site (Shaferman *et al.*, 1992). They are thus likely to be related to the multiple effects of the various peripheral ligands, including ACh in excess, on active center reactivity (Pattison and Bernhard, 1978; Tomlinson *et al.*, 1980; Berman and Leonard, 1990). Fasciculins, the three-dimensional structure of which is now solved (Le Du *et al.*, 1992), are too large to enter the gorge, except with severe writhing. Not to negate the hypothesis of steric hindrance, fasciculins should therefore mask the gorge entrance by binding to its rim. Thus, fasciculin occupation of its own peripheral binding site would preclude crossing of ACh, as well as of catalytic site-directed inhibitors, through the gorge. Alternatively, fasciculins may sufficiently perturb the structure of AChE to be able to inhibit the substrate catalysis allosterically. AChE inhibition would thus result from structural modification of the catalytic site or/and disturbance of the postulated guidance mechanism for ACh. Actually, bound fasciculin fails to prevent subsequent occupation of the esteratic subsite of AChE catalytic site, since the total number of [1,3-³H]DFP binding sites, as well as the kinetics of [1,3-³H]DFP association, was found unchanged after preincubation of P₂ with FAS3 (Fig. 8B). This result evidences that a ternary complex FAS3·AChE·DFP may form with a 1:1:1 ratio and, thus, invalidates the hypothesis of steric hindrance. Since phosphorylation, by DFP, of the AChE catalytic serine can still occur on the FAS3·AChE complex, the AChE inhibition due to fasciculin tight binding

may be thought to proceed from structural modification, at a distance, of the anionic subsite of the AChE catalytic site. Whether this subsite is actually modified should be elucidated by resolution of the FAS·AChE-complex x-ray structure.

*Acknowledgments*—We thank Dr. M. F. Martin-Eauclaire and B. Céard for chromatographic help and advice, T. Brando for amino acid analyses, and M. Alvitre for rat care. We are particularly grateful to Dr. J. Massoulié for the gift of MPT, to Dr. A. Cornish-Bowden for fruitful discussion, and to Prof. H. Rochat for interest and support.

## REFERENCES

Aldridge, W. N., and Reiner, E. (1969) *Biochem. J.* **115**, 147-162

Anderson, A. J., Harvey, A. L., and Mbugua, P. M. (1985) *Neurosci. Lett.* **54**, 123-128

Augustinsson, K. B. (1963) in *Cholinesterases and Anticholinesterase Agents, Handbook of Experimental Pharmacology*. (Koelle, G. B., ed) Vol. 15, pp. 89-128, Springer-Verlag, Berlin

Austin, L., and Berry, W. K. (1953) *Biochem. J.* **54**, 695-700

Bechis, G., Granier, C., Van Rietschoten, J., Jover, E., Rochat, H., and Miranda, F. (1976) *Eur. J. Biochem.* **68**, 445-456

Bechis, G., Sampieri, F., Yuan, P.-M., Brando, T., Martin, M. F., Diniz, C. R., and Rochat, H. (1984) *Biochem. Biophys. Res. Commun.* **122**, 1146-1153

Berman, H. A., and Leonard, K. (1990) *Biochemistry* **29**, 10640-10649

Berman, H. A., and Taylor, P. (1978) *Biochemistry* **17**, 1704-1713

Berman, H. A., Becktel, W., and Taylor, P. (1981) *Biochemistry* **20**, 4803-4810

Bisso, G. M., Biancesco, R., and Michaelek, H. (1991) *Neurochem. Res.* **16**, 571-575

Boynaems, J. M., and Dumont, J. E. (1980) *Outlines of Receptor Theory* (Boynaems, J. M., and Dumont, J. E., eds) pp. 5-14, Elsevier/North-Holland Medical Press, Amsterdam

Bougis, P. E., Tessier, M., Van Rietschoten, J., Rochat, H., Faucon, J. F., and Dufourcq, J. (1983) *Mol. Cell. Biochem.* **55**, 49-64

Bougis, P. E., Marchot, P., and Rochat, H. (1986) *Biochemistry* **25**, 7235-7243

Cervensansky, C., Dajas, F., Harvey, A. L., and Karlsson, E. (1991a) in *Snake Toxins* (Harvey, A. L., ed) pp. 303-321, Pergamon Press, Inc., New York

Cervensansky, C., Engström, A., and Karlsson, E. (1991b) *Toxicon* **29**, 1163-1164

Chang, J. Y. (1983) *Methods Enzymol.* **91**, 455-466

Changeux, J. P. (1966) *Mol. Pharmacol.* **2**, 369-392

Cohen, J. A., and Oosterbaan, R. A. (1963) in *Cholinesterases and Anticholinesterase Agents Handbook of Experimental Pharmacology* (Koelle, G. B., ed) Vol. 15, pp. 299-373, Springer-Verlag, Berlin

Du cancel, F., Bouchier, C., Tamiya, T., Boulain, J. C., and Ménez, A. (1991) in *Snake Toxins* (Harvey, A. L., ed) pp. 395-414, Pergamon Press, Inc., New York

Ellman, G. L., Courtney, K. D., Andres, Jr. V., and Featherstone, R. M. (1961) *Biochem. Pharmacol.* **7**, 88-95

Epstein, D. J., Berman, H. A., and Taylor, P. (1979) *Biochemistry* **18**, 4749-4754

Faure, G., Boulain, J. C., Bouet, F., Montenay-Garestier, T., Fromageot, P., and Ménez, A. (1983) *Biochemistry* **22**, 2068-2076

Fishbein, J. C., Place, A. R., Ropson, I., Powers, D. A., and Sofer, W. (1980) *Anal. Biochem.* **108**, 193-201

Friboulet, A., Goudou, D., and Rieger, F. (1986) *Neurochem. Int.* **9**, 323-328

Friboulet, A., Rieger, F., Goudou, D., Amitai, G., and Taylor, P. (1990) *Biochemistry* **29**, 914-920

Gatineau, E., Takechi, M., Bouet, F., Mansuelle, P., Rochat, H., Harvey, A. L., Montenay-Garestier, Th., and Menez, A. (1990) *Biochemistry* **29**, 6480-6489

Gray, E. G., and Whittaker, V. P. (1962) *J. Anat.* **96**, 79-88

Gross, E. (1967) *Methods Enzymol.* **11**, 238-241

Harvey, A. L., Anderson, A. J., Mbugua, P. M., and Karlsson, E. (1984) *J. Toxicol. Toxin Rev.* **3**, 91-137

Houmad, J., and Drapeau, G. R. (1972) *Proc. Natl. Acad. Sci. U. S. A.* **69**, 3506-3509

Joubert, F. J., and Taljaard, N. (1978) *South Afr. J. Chem.* **31**, 107-110

Karlsson, E., Mbugua, P. M., and Rodriguez-Ithurralde, D. (1984) *J. Physiol. (Paris)* **79**, 232-240

Kopeyan, C., Miranda, F., and Rochat, H. (1975) *Eur. J. Biochem.* **58**, 117-122

Le Du, M. H., Marchot, P., Bougis, P. E., and Fontecilla-Camps, J. C. (1989) *J. Biol. Chem.* **264**, 21401-21402

Le Du, M. H., Marchot, P., Bougis, P. E., and Fontecilla-Camps, J. C. (1992) *J. Biol. Chem.* **267**, 22122-22130

Lin, W. W., Lee, C. Y., Carlsson, F. H. H., and Joubert, F. J. (1987) *Asia Pac. J. Pharmacol.* **2**, 79-85

Long, J. P. (1963) in *Cholinesterases and Anticholinesterase Agents Handbook of Experimental Pharmacology* (Koelle, G. B., ed) Vol. 15, pp. 374-427, Springer-Verlag, Berlin

Marchot, P. (1986) *Contribution à l'Etude de la Purification et du Mode d'Action des Toxines des Venins de Serpents Elapidae*. Thèse de Doctorat de l'Université d'Aix-Marseille II (Neurosciences)

Marchot, P., Frachon, P., and Bougis, P. E. (1988) *Eur. J. Biochem.* **174**, 537-542

Martin, B. M., Chibber, B. A., and Maelicke, A. (1983) *J. Biol. Chem.* **258**, 8714-8722

Martin, M. F., Garcia y Perez, L. G., El Ayeb, M., Kopeyan, C., Bechis, G., Jover, M., and Rochat, H. (1987) *J. Biol. Chem.* **262**, 4452-4459

Massoulié, J., and Bon, S. (1982) *Annu. Rev. Neurosci.* **5**, 57-106

Moserer, G., and Sigman, D. S. (1974) *Biochemistry* **13**, 2299-2307

Murphy, S. D., Coster, L. G., and Wang, C. (1984) *Cell. Mol. Neurotoxicol.* **165**, 176-179

Neurath, H. (1984) *Science* **224**, 350-357

Pattison, S., and Bernhard, S. (1978) *Proc. Natl. Acad. Sci. U. S. A.* **75**, 3613-3617
Puu, G., and Koch, M. (1990) *Biochem. Pharmacol.* **40**, 2209-2214

Quinn, D. M. (1987) *Chem. Rev.* **87**, 955-979

Radic, Z., Reiner, E., and Taylor, P. (1991) *Mol. Pharmacol.* **39**, 98-104

Rieger, F., and Vigny, M. (1976) *J. Neurochem.* **27**, 121-129

Robaire, B., and Kato, G. (1974) *Biochem. Pharmacol.* **23**, 2476-2480

Rochat, H., Tessier, M., Miranda, F., and Lissitzky, S. (1977) *Anal. Biochem.* **82**, 532-548

Rodriguez-Ithurralde, D., Silveira, R., Barbeito, L., and Dajas, F. (1983) *Neurochem. Int.* **5**, 267-274

Rosenberry, T. L. (1975) in *Advances in Enzymology* (Meister, A., ed) Vol. 43, pp. 103-218, Interscience, New York

Sato, S., and Tamiya, N. (1970) *J. Biochem. (Tokyo)* **68**, 867-872

Shafferman, A., Velan, B., Ordentlich, A., Kronman, C., Grosfeld, H., Leitner, M., Flashner, Y., Cohen, S., Barak, D., and Ariel, N. (1992) *EMBO J.* **11**, 3561-3568

Sindhuphak, R., Karlsson, E., Conradi, S., and Ronnevi, L. O. (1988) *J. Neurol. Sci.* **86**, 195-202

Sussman, J. L., Harel, M., Frolow, F., Oefner, C., Goldman, A., Toker, L., and Silman, I. (1991) *Science* **253**, 872-879

Taylor, P., and Lappi, S. (1975) *Biochemistry* **14**, 1989-1997

Tomlinson, G., Mutus, B., and McLennan, I. (1980) *Mol. Pharmacol.* **18**, 33-39

Vigny, M., Bon, S., Massoulié, J., and Leterrier, F. (1978) *Eur. J. Biochem.* **85**, 317-323

Viljoen, C. C., and Botes, D. P. (1973) *J. Biol. Chem.* **248**, 4915-4919

Weber, M., and Changeux, J. P. (1974a) *Mol. Pharmacol.* **10**, 1-14

Weber, M., and Changeux, J. P. (1974b) *Mol. Pharmacol.* **10**, 15-34

Zorko, M., and Pavlic, M. (1986) *Biochem. Pharmacol.* **35**, 2287-2296