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The Role of Zinc Binding in the Biological Activity of Botulinum Toxin

2001; Elsevier BV; Volume: 276; Issue: 29 Linguagem: Inglês

10.1074/jbc.m102172200

1083-351X

Lance L. Simpson, Andrew B. Maksymowych, Sheryl Hao,

Cellular transport and secretion

Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn2+ from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn2+. In contrast to soluble chelators, immobilized chelators have no effect on bound Zn2+, nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn2+ from its coordination site in botulinum toxin is relatively slow. When exogenous Zn2+ is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn2+ can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn2+ does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn2+ or exogenous Zn2+ means that productive internalization does not produce conformational changes that block rebinding of cation. Botulinum toxin is a zinc-dependent endoprotease that acts on vulnerable cells to cleave polypeptides that are essential for exocytosis. To exert this poisoning effect, the toxin must proceed through a complex sequence of events that involves binding, productive internalization, and intracellular expression of catalytic activity. Results presented in this study show that soluble chelators rapidly strip Zn2+ from its binding site in botulinum toxin, and this stripping of cation results in the loss of catalytic activity in cell-free or broken cell preparations. Stripped toxin is still active against intact neuromuscular junctions, presumably because internalized toxin binds cytosolic Zn2+. In contrast to soluble chelators, immobilized chelators have no effect on bound Zn2+, nor do they alter toxin activity. The latter finding is because of the fact that the spontaneous loss of Zn2+ from its coordination site in botulinum toxin is relatively slow. When exogenous Zn2+ is added to toxin that has been stripped by soluble chelators, the molecule rebinds cation and regains catalytic and neuromuscular blocking activity. Exogenous Zn2+ can restore toxin activity either when the toxin is free in solution on the cell exterior or when it has been internalized and is in the cytosol. The fact that stripped toxin can reach the cytosol means that the loss of bound Zn2+ does not produce conformational changes that block internalization. Similarly, the fact that stripped toxin in the cytosol can be reactivated by ambient Zn2+ or exogenous Zn2+ means that productive internalization does not produce conformational changes that block rebinding of cation. tetrakis-(2-pyridylmethyl) ethylenediamine glutathioneS-transferase synaptosomal protein of 25 kDa botulinum neurotoxin serotype A Botulinum toxin poisons vulnerable cells by proceeding through a sequence of three major steps: binding, productive internalization, and intracellular expression of catalytic activity (1Humeau Y. Doussau F. Grant N.J. Poulain B. Biochimie (Paris). 2000; 85: 427-446Crossref Scopus (388) Google Scholar). Each of the three major steps in toxin action can be further subdivided into two or more events. Thus, the binding step reportedly involves two sequential processes. During the first, toxin associates with low affinity receptors on the cell surface, and during the second, the low affinity complex shuttles the toxin to a high affinity receptor (2Montecucco C. Trends Biochem. Sci. 1986; 11: 315-317Abstract Full Text PDF Scopus (349) Google Scholar). Productive internalization also involves a number of events (see "Discussion"), with the two major ones being receptor-mediated endocytosis across the plasma membrane and pH-dependent translocation across the endosome membrane. During the final step, botulinum toxin acts as a zinc-dependent endoprotease to cleave polypeptides that govern exocytosis (3Montecucco C. Schiavo G. Q. Rev. Biophys. 1995; 28: 423-472Crossref PubMed Scopus (409) Google Scholar). Enzymatic cleavage of these polypeptides must also involve multiple events (viz. substrate binding, substrate cleavage), although this has not been studied extensively. During the interval since toxin action was first described as a sequence of three steps (4Simpson L.L. J. Pharmacol. Exp. Ther. 1980; 212: 16-21PubMed Google Scholar, 5Simpson L.L. Pharmacol. Rev. 1981; 33: 155-188PubMed Google Scholar), investigators have tended to examine these steps as though they are separate and independent. However, recent and apparently contradictory findings may necessitate a change in perspective. In 1992, Schiavo et al. (6Schiavo G. Benfanti F. Poulain B. Rossetto O. Polverino de Laureto P. DasGupta B. Montecucco C. Nature. 1992; 359: 832-835Crossref PubMed Scopus (1495) Google Scholar) and Linket al. (7Link E. Edelmann L. Chou J.H. Binz T. Yamasaki S. Eisel U. Baumert M. Sudhof T.C. Niemann H. Jahn R. Biochem. Biophys. Res. Commun. 1992; 189: 1017-1023Crossref PubMed Scopus (268) Google Scholar) reported that botulinum toxin type B and tetanus toxin were zinc-dependent endoproteases. In 1993, Simpsonet al. (8Simpson L.L. Coffield J.A. Bakry N. J. Pharmacol. Exp. Ther. 1993; 267: 720-727PubMed Google Scholar) reported that all seven botulinum toxin serotypes (A–G) were likely to be zinc-dependent endoproteases, because the toxicity of all seven serotypes was diminished by zinc chelation. However, the work with Zn2+ chelators revealed an unexpected outcome. It was observed that removing Zn2+from toxin was necessary but not sufficient to cause a loss of toxicity against intact cells. Toxin stripped of Zn2+ could still produce blockade of exocytosis, apparently because the toxin could replenish its divalent cation from tissue stores. In keeping with this idea, the chelation of toxin as well as tissue was necessary to produce marked reductions in toxin activity. More recently, Fu et al. (9Fu F.-N. Lomneth R.B. Cai S. Singh B.R. Biochemistry. 1998; 37: 5267-5278Crossref PubMed Scopus (64) Google Scholar) have published findings that seem to contradict those just described. They found that the use of a chelator to remove Zn2+ from toxin led to irreversible changes in tertiary structure as measured by various light-scattering techniques. They also found that removal of the cation led to irreversible losses in toxicity as measured by norepinephrine release from permeabilized PC-12 cells. Close inspection of these contradictory findings on Zn2+reveals that there could be implications that extend beyond the final or catalytic step in toxin action. These data may also have an impact on proposed models for productive internalization and particularly on models for toxin penetration of the endosome membrane (see "Discussion"). Thus, any model that calls for a significant relaxation of the toxin molecule, which in turn could cause the loss of Zn2+ binding, would not be plausible unless the toxin could subsequently regain Zn2+ and biological activity. Conversely, any model that calls for toxin to remain tightly bound to Zn2+ during translocation would not be viable unless the translocation process could accommodate a somewhat bulky protein that retains at least some of its secondary and tertiary structure. In the work that follows, both experimental studies and deduction have been used to gauge the likelihood that removal of Zn2+produces irreversible losses in botulinum toxin activity. This work then has been used to assess whether events that occur during the internalization step can impact events during the catalytic step. 65ZnCl2 (6,146.68 MBq/mg) was purchased from PerkinElmer Life Sciences. Sephadex G-25 gel filtration columns were obtained from Amersham Pharmacia Biotech. Chelex® 100 Resin was purchased from Bio-Rad. EGTA and EDTA disodium salts were from Sigma, and TPEN1 was purchased from Molecular Probes. Rabbit anti-C-terminal SNAP-25 polyclonal antibody was purchased from StressGen Biotechnologies Corp. (Victoria, B.C., Canada), and donkey anti-rabbit IgG horseradish peroxidase was fromAmersham Pharmacia Biotech. All other reagents were from Sigma or Fisher Scientific (Malvern, PA). Botulinum neurotoxin type A was purified to homogeneity by procedures that have been described previously (10Sakaguchi G. Pharmacol. Ther. 1982; 19: 165-194Crossref PubMed Scopus (300) Google Scholar, 11DasGupta B.R. Sathyamoorthy V. Toxicon. 1984; 22: 415-424Crossref PubMed Scopus (127) Google Scholar, 12Simpson L.L. Schmidt J.J. Middlebrook J.L. Methods Enzymol. 1988; 165: 76-85Crossref PubMed Scopus (23) Google Scholar). Recombinant GST-SNAP-25 (∼50 kDa) was constructed and expressed according to published techniques. Murine SNAP-25 cDNA, pSNAP8.52, which was derived from a BALB/c strain brain Lambda cDNA library (13Oyler G.A. Higgins G.A. Hart R.A. Battenberg E. Billingsley M. Bloom F.E. Wilson M.C. J. Cell Biol. 1989; 109: 3039-3052Crossref PubMed Scopus (704) Google Scholar) kindly provided by Dr. Michael C. Wilson (Scripps Clinic, La Jolla, CA), was used as a polymerase chain reaction template. The polymerase chain reaction was carried out with primers 5′-TCT TGG ATC CGC CGA AGA CGC AGA CAT GC-3′ and 5′-TCT TGG ATC CTT AAC CAC TTC CCA GCA TCT T-3′. A product of ∼880 base pairs was digested with BamHI and ligated into pGEX-KG vector (14Guan K.L. Dixon J.E. Anal. Biochem. 1991; 192: 262-267Crossref PubMed Scopus (1700) Google Scholar). The resulting construct was sequenced to confirm that SNAP-25 cDNA had been inserted into pGEX-KG correctly. The transformation, expression of pGEX-KG/SNAP-25 in bacteria, and purification of GST-SNAP-25 fusion protein were performed as described (15Wong S.H. Zhang T. Xu Y. Subramaniam V.N. Griffiths G. Hong W. Mol. Biol. Cell. 1998; 9: 1549-1563Crossref PubMed Scopus (100) Google Scholar). All experiments were performed using buffers that were pretreated with Chelex®100 resin to remove trace amounts of cations such as Zn2+. Calculations based on manufacturer specifications indicated that 0.5 mg of Chelex® 100 resin/liter of buffer should remove all trace cations from solution. The buffers used in these experiments were pretreated with a substantial excess of resin (5 g/liter) for 18 h at room temperature, and this was followed by filtration through a 0.2-µm filter to remove the resin. Prior to use, all filters and labware were rinsed three times with Chelex®100-treated distilled water. For all experimental methods described below, 65Zn associated with toxin was quantified using Sephadex G-25 chromatography followed by liquid scintillation counting. Columns were equilibrated by washing with a column buffer (50 mm NaPO4 and 1 m NaCl, pH 6.8). Subsequently, 0.5-ml experimental samples of toxin were filtered through Sephadex G-25 columns, and ∼0.5-ml (10-drop) fractions were collected. The samples were added to 3 ml of scintillation fluid, and the amount of radioactivity in the fractions was determined by liquid scintillation spectrometry. Labeled toxin eluted at void volume, and the radioactivity contained in the void-volume fractions was summed to determine the total amount of 65Zn label associated with the toxin peak. Zn2+ depletion of toxin was carried out in 150 mm Tris-HCl buffer, pH 7.4, employing two steps. First, toxin was added to buffer containing 10 mm EDTA and EGTA (final concentrations) and incubated at 37 °C for 1 h. Next, the toxin sample was dialyzed overnight at 3 °C (18 h, minimum of two buffer changes) against this same buffer containing 10 mm EDTA and EGTA. After Zn2+depletion, the toxin sample was dialyzed into 50 mm sodium phosphate buffer, pH 7.0, that had been pretreated with Chelex® 100 resin for 24 h (two buffer changes). Stripped (e.g. Zn2+-depleted) toxin was stored at 3 °C in sterile vials that had been rinsed three times with Chelex®-treated buffer, and it was used over a period of ∼6 months. For Zn2+ exchange, pure BoNT/A (1 × 10−7m final concentration) and 65ZnCl2(10−8–10−5m final concentration) were incubated in a 500-µl reaction volume in Chelex® 100-treated buffer (100 mm Tris-HCl and 50 mm NaCl, pH 7.2) at room temperature for 24 h. For Zn2+ replacement, Zn2+-depleted BoNT/A (1 × 10−7m final concentration) and65ZnCl2 (2 × 10−7m final concentration) were incubated in a 500-µl reaction volume in Chelex® 100-treated buffer (100 mm Tris-HCl and 50 mm NaCl, pH 7.2) at room temperature for 1, 5, 15, 45, 60, and 120 min. After incubation, the65Zn label associated with the toxin peak was determined as described above. Zn2+-depleted BoNT/A (1 × 10−7m final concentration) was prelabeled with 65Zn (2 × 10−7m final concentration) for 18 h at 3 °C in reaction buffer. The experiment with prelabeled toxin was initiated by warming the materials to room temperature and subsequently adding 30 mg of Chelex® 100 resin, EDTA (30 mm final concentration), or TPEN (20 µm final concentration) to 500-µl reaction vessels. Control reactions used prelabeled toxin without added chelator. Experimental samples were incubated at room temperature for varying lengths of time (see "Results"), after which the toxin-associated 65Zn label was determined. Zn2+-depleted BoNT/A (1 × 10−7m final concentration) was prelabeled with 65Zn (2 × 10−7m final concentration) as described above. The experiment with prelabeled toxin was initiated by warming to room temperature and adjusting the 500-µl reaction mixture to a pH of ∼4.00, ∼5.00, or ∼6.00 by the addition of the appropriate amount of 1 n HCl. Control reactions remained at pH 7.20. Experimental samples were incubated at room temperature for 30 min. For each sample, the 65Zn label associated with the toxin peak was determined. Brain tissue was removed from mice and immediately immersed and homogenized in chilled buffer containing 255 mm sucrose, 1 mm EDTA, and 20 mm Hepes, pH. 7.4. The resulting suspension was homogenized with a Brinkmann homogenizer (E2M/Polytron, Norcross, GA) at a setting of ∼4–5 for ∼1 min. The homogenates were centrifuged at 3,000 × g for 5 min (SS-34 Rotor, Sorvall RC-5B Refrigerated Superspeed centrifuge). The resulting supernatant (S1) was centrifuged at 10,000 × g for 10 min (same rotor and centrifuge). The second supernatant (S2) was centrifuged at 250,000 × g for 70 min (T-865.1 Rotor, Sorvall Ultra 80 centrifuge). The resulting pellet (P3) was resuspended in 50 mm Hepes, pH 7.1. The protein concentration of P3 was determined using a Bio-Rad protein assay kit (Bio-Rad). BoNT/A was assayed for endoprotease activity using either recombinant GST-SNAP-25 or synaptosomal SNAP-25. In both cases, an anti-C-terminal SNAP-25 antibody was used as a reagent in the immunodetection of enzyme activity (16Poirier M. Hao J. Malkus P. Chan C. Moore M. King D. Bennett M. J. Biol. Chem. 1998; 273: 11370-11377Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar). When recombinant substrate was used, ∼130 ng of GST-SNAP-25 was present in each reaction vessel; when synaptosomes were used, 7–10 µg of P3 protein was added to each reaction. In the latter case, 1%N-octyl-β-d-glucopyranoside was added to the reaction to permeabilize synaptosomes and expose SNAP-25. Reactions were initiated by reducing BoNT/A (100–200 nm) with dithiothreitol (8 mm) at room temperature for 30 min in cleavage buffer (50 mm Hepes, pH 7.1, plus 20 µm ZnCl2). When the assays were done with stripped toxin or chelators, auxiliary Zn2+ was not added to the reactions mixtures. In most experiments, substrate was added to reduced toxin, and the reaction was allowed to proceed for 3 h at 37 °C. Enzymatic cleavage reactions were terminated by adding an equal volume of 2× sample buffer (125 mm Tris-HCl, pH 6.8, 1% SDS, 20% glycerol, 700 mm β-mercaptoethanol, and 0.01% bromphenol blue). The samples were denatured by incubating at 95 °C with subsequent cooling and then separated in 10% polyacrylamide gels according to Laemmli (17Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (214196) Google Scholar). SNAP-25 was transferred from gels to nitrocellulose membranes (0.45 µm, Immobilon-NC and Fisher Scientific) and probed with anti-SNAP-25 C-terminal antibody at 1:10,000 dilution. Donkey anti-rabbit IgG conjugated with horseradish peroxidase was used as a secondary antibody at a dilution of 1:10,000. The antibody reaction was visualized by chemiluminescence (ECL Plus, Amersham Pharmacia Biotech) using x-ray film (Kodak Bio-Max). Phrenic nerve hemidiaphragms were excised from mice as described previously (18Coffield J.A. Bakry N. Zhang R.D. Carlson J. Gomella L.G. Simpson L.L. J. Pharmacol. Exp. Ther. 1997; 280: 1489-1498PubMed Google Scholar, 19Kiyatkin N. Maksymowych A.B. Simpson L.L. Infect. Immun. 1997; 65: 4586-4591Crossref PubMed Google Scholar). The tissues were suspended in physiological solution that was bubbled with 95% O2 and 5% CO2 and maintained at 35 °C. Unless otherwise indicated, the physiological solution had the following composition: 137 mm NaCl, 5 mm KCl, 1.8 mm CaCl2, 1.0 mmMgSO4, 24 mm NaHCO3, 1.0 mm NaH2PO4, and 11 mm d-glucose. The solutions were supplemented with 0.01% gelatin as an auxiliary protein to diminish nonspecific inactivation of toxin. Phrenic nerves were stimulated continuously (0.5 Hz, 0.2-msec duration), and muscle twitch was recorded. Toxin-induced paralysis was measured as a 90% reduction in muscle twitch response to nerve stimulation. Two paradigms were used to expose BoNT/A to soluble chelators. In the first, toxin was exposed for lengthy periods of time to a combination of EDTA and EGTA to strip Zn2+ (see "Experimental Procedures"). In the second, toxin was incubated with TPEN for 60 min at room temperature before the assays. For both protocols, endoprotease activity was determined using recombinant substrate (GST-SNAP-25) or endogenous substrate (synaptosomal SNAP-25). As shown in Fig. 1, native toxin produced complete cleavage of substrate (A–D, lane 2 in each panel). By contrast, toxin that had been incubated with TPEN (A and C, lane 3 in each panel) and toxin that had been stripped of Zn2+ (B andD, lane 3 in each panel) were essentially free of enzymatic activity. Interestingly, the addition of exogenous Zn2+ reversed the effects of chelation. Regardless of whether toxin had been transiently exposed to TPEN (A andC, lane 4 in each panel) or stripped of Zn2+ (B and D, lane 4 in each panel), the addition of micromolar amounts of Zn2+restored endoprotease activity. Samples of toxin that had been stripped of Zn2+ with EDTA and EGTA or had been exposed transiently to TPEN were bioassayed for their abilities to produce neuromuscular blockade of phrenic nerve hemidiaphragm preparations (Fig. 2). In both cases, treated toxin proved to be highly effective in blocking exocytosis. This result was obtained even though chelator-treated toxin was not exposed to exogenous Zn2+ before its addition to tissues. Experiments were done with Chelex®, an immobilized chelator that cannot gain access to Zn2+ within its coordination site in the toxin molecule but can bind free Zn2+ in solution. The toxin was incubated with chelator as described under "Experimental Procedures" for 120 min at room temperature, after which the mixture was centrifuged to separate the components. Soluble toxin was then assayed for biological activity. Interestingly, toxin exposed to Chelex® possessed enzymatic activity and neuromuscular blocking activity comparable with those of native untreated toxin (e.g. Fig. 1,A–D, lane 2; Fig. 2). These results were observed even without the addition of exogenous Zn2+(results not illustrated). Experiments were done to determine the rate of spontaneous exchange of zinc at the active site of botulinum toxin. For this purpose, four concentrations of 65Zn (10−8–10−5) were incubated with a single concentration of BoNT/A (1 × 10−7m). Experimental samples were incubated for 24 h at room temperature. Subsequently, the amount of exogenous radioactive Zn2+ that replaced endogenous unlabeled Zn2+ was determined. As shown in Fig. 3 A, there was concentration-dependent replacement of endogenous Zn2+. An exchange was evident even at 10−7m and was more pronounced at 10−6 and 10−5m. These results are consistent with the previously reported finding that the apparent K d for Zn2+ at its binding site is ∼60–80 nm(20Schiavo G. Rossetto O. Santucci A. DasGupta B.R. Montecucco C. J. Biol. Chem. 1992; 267: 23479-23483Abstract Full Text PDF PubMed Google Scholar). In a subsequent set of experiments, the precise time course of Zn2+ exchange was studied. This was done at a65Zn concentration of 2 × 10−7m, which is on the linear portion of the concentration-effect curve shown in Fig. 3 A. The results indicated that a steady state for exchange was attained within ∼5–6 days, and the half-time for exchange was ∼2–3 days (Fig.3 B). This means that when studied in physiological buffer at pH 7.2, endogenous Zn2+ can exchange with environmental Zn2+. The time course of this exchange is such as to make it possible for botulinum toxin molecules to undergo at least a fractional exchange of Zn2+ during their lifetime (e.g. synthesis and release by bacteria, ingestion and absorption by victim, and binding, internalization, and onset of neuromuscular blockade). The results presented above and illustrated in Fig. 1strongly suggest that stripped toxin can regain Zn2+ and enzymatic activity. Furthermore, the in vitro toxicity experiments with phrenic nerve hemidiaphragm preparations indicate that stripped toxin can accumulate Zn2+ rapidly (Fig. 2). Therefore, experiments were done to quantify the rate at which Zn2+ can be acquired. Native toxin was stripped of endogenous Zn2+ with a combination of EDTA and EGTA as described under "Experimental Procedures." Stripped toxin (1 × 10−7m) was then incubated for various amounts of time with65Zn (2 × 10−7m), and the rate of incorporation of exogenous zinc was monitored. The data, which are shown in Fig. 4, indicate that the half-time for accumulation was ∼6 min. This finding supports two conclusions: (a) the rate of Zn2+ accumulation by stripped toxin is much greater than the rate of Zn2+exchange in native toxin, and (b) the rate of Zn2+ accumulation by stripped toxin can easily account for the ability of this material to acquire exogenous Zn2+ and block neuromuscular transmission (e.g. Fig. 2). Bioassays for toxin activity in cleaving substrate and in blocking neuromuscular transmission have shown that a soluble chelator such as EDTA abolishes toxicity (Fig. 1), whereas an immobilized chelator such as Chelex® 100 does not (see above). This suggests that within the finite time course of an experiment, EDTA efficiently strips Zn2+, but Chelex® 100 does not. Therefore, experiments were done to test this premise. Botulinum toxin was stripped of endogenous Zn2+ as described under "Experimental Procedures" and then loaded with65Zn. This material was then incubated with EDTA (30 mm), TPEN (20 µm), or Chelex®for 3 h at room temperature, after which the residual65Zn associated with the toxin was quantified. The averaged data from three experiments showed that Chelex® had no detectable ability to strip Zn2+ from toxin. On the other hand, EDTA removed more than 98% of bound Zn2+, and TPEN removed ∼89% of the bound cation. These findings indicate that soluble chelators act rapidly to remove Zn2+ from toxin. Furthermore, the results indicate that soluble chelators do more than merely bind Zn2+. In all likelihood, they approach the active site and scavenge bound Zn2+. And finally, the data showing that Chelex® is not very effective in stripping Zn2+ accounts for the finding that Chelex® is not effective in abolishing toxicity (see above). Botulinum toxin is not likely to encounter a chelator when it acts on neuromuscular junctions to paralyze transmission. However, there are ambient conditions encountered by the toxin that could produce a loss of Zn2+. The most notable of these is the fall in pH that is encountered when the toxin translocates from the lumen of the endosome to the cytosol. The fall in pH could produce a loss of Zn2+because low pH leads to protonation of histidine residues that coordinate Zn2+ binding, low pH produces conformational changes that diminish Zn2+ binding, or both. Therefore, experiments were done to quantify Zn2+ retention at pH values (4.0–7.2) that bridge the pK a for the imidazole side chain of histidine residues in proteins (6.1–6.4). Coincidentally, these pH values also bridge the range for those that induce conformational changes associated with toxin translocation. Botulinum toxin was stripped of Zn2+ as described above and loaded with 65Zn. This material was then incubated with solutions of varying pH for 30 min at room temperature. When the levels of residual 65Zn were quantified, the results (Fig. 5) showed that there was a significant loss of cation at a pH of 6.0. There was an additional loss of cation with further lowering of pH. The exact pH at which toxin is induced to translocate from the endosome to the cytosol has not been determined. Nevertheless, the pH of primary endosomes is in the range of 6.2–6.3, and the pH of later stage endosomes is even lower (21Mostov K.E. Verges M. Altschuler Y. Curr. Opin. Cell Biol. 2000; 12: 483-490Crossref PubMed Scopus (336) Google Scholar). Therefore, the fact that there is a significant loss of Zn2+ at pH 6.0 suggests that, at least potentially, some endogenous cation could be lost during translocation. This could be caused by protonation of histidines, change in conformation, or both. As reported previously, pretreatment of toxin with a soluble chelator is not sufficient to cause the molecule to lose neuromuscular blocking activity (Ref. 8Simpson L.L. Coffield J.A. Bakry N. J. Pharmacol. Exp. Ther. 1993; 267: 720-727PubMed Google Scholar; see above). Stripped toxin can regain Zn2+ from tissue stores and thus regain catalytic and neuromuscular blocking activity. A loss of activity is observed only if both toxin and tissue are pretreated with the chelator. Fig. 6 illustrates the results of an experiment in which native toxin and TPEN-pretreated toxin (10 µm) were added either to control tissues or to TPEN-pretreated tissues (10 µm). These data clearly show that native toxin and stripped toxin were comparable in their abilities to block neuromuscular transmission in control tissues (Aand B). They also show that neither native toxin nor stripped toxin blocked transmission when added to tissues incubated in TPEN (A and B). In a subsequent set of experiments, native toxin and stripped toxin were added to tissues pretreated with TPEN. After an elapsed time equivalent to that necessary for toxin to paralyze control tissues (i.e. Fig. 6 A), a molar excess of Zn2+ (20 µm) was added to preparations (C). It is noteworthy that (a) there was early onset of toxin action that eventually culminated in neuromuscular blockade, and (b) there was no significant difference between native toxin and stripped toxin in terms of rate of onset of blockade. The results in Fig. 6 C suggest that paralysis was caused by toxin that had already undergone productive internalization. To confirm this hypothesis, tissues were treated identically to that for tissues in C except that tissues were washed several times prior to the addition of Zn2+. Thus, there was no toxin left in solution for binding and later internalization. When tissues were washed free of extracellular toxin and then exposed to Zn2+, there was again a rapid onset of toxin action (D). Interestingly, there was no difference between tissues that were continuously exposed to toxin (C) and those that were washed to remove unbound toxin (D). The data presented above show that soluble, but not immobilized, chelators can strip Zn2+ from toxin. Stripped toxin has little ability to cleave substrate or to block neuromuscular transmission. However, toxin that regains Zn2+ from tissue stores or from external sources regains biological activity. The data in Fig. 6, and particularly those on control toxin added to TPEN-pretreated tissue, presents a special circumstance. The fact that native toxin is rendered inactive when added to tissues incubated in TPEN suggests that a soluble chelator can strip the toxin in less time than that needed for the toxin to enter the nerve and begin cleaving substrate. This premise was tested by examining the rate of TPEN-induced loss of Zn2+. Samples of toxin (1 × 10−7m) were stripped and reloaded with 65Zn as described above. This material was incubated with TPEN (2 × 10−5m) for varying lengths of time, after which zinc retention was measured. As shown in Fig.7, TPEN produced a significant loss of zinc within 20 min, and the loss continued to occur for at least 180 min. These data provide qualitative support for the premise that TPEN can strip Zn2+ during the brief interval when toxin is en route to its site of action. Indeed, the actual sequence of events in the tissue bath is likely to be even more dramatic than that suggested by Fig. 7. For the Zn2+ stripping experiment (e.g.Fig. 7) the ratio of chelator to toxin was ∼200 (2 × 10−5m TPEN; 1 × 10−7m toxin). For the neuromuscular blocking experiments, the ratio was 2,000,000 (2 × 10−5m TPEN; 1 × 10−11m toxin). Therefore, the actual rate of Zn2+ stripping while the toxin is en route to its site of actio