Biochemical and physiological properties of a purified snake venom neurotoxin which acts presynaptically

JOURNAL OF NEUROBIOLOGY, VOL. 5, NO. 2, PP. 135-150

BIOCHEMICAL AND PHYSIOLOGICAL PROPERTIES OF A PURIFIED SNAKE VENOM NEUROTOXIN WHICH ACTS

PRESYNAPTICALLY

REGIS B. KELLY and FRANK R. BROWN III*

Department of Biochemistry and Biophysics, University of California, San Francisco, Sun Francisco, California 94122

SUMMARY

A highly toxic protein has been isolated from the venom of the snake Bungurus multicinctus. No major contaminants were detected on isoelectric focusing, gel filtration, or gel electrophoresis. The molecular weight calculated from its amino acid composition (21,800) agreed well with that calculated from gel filtration and equilibrium sedimentation data. Gel electrophoresis showed that the toxin consisted of two subunits of molecular weight 8,800 and 12,400 held together by disulfide bonds. Both the structure, determined by circular dichroism, and the biological activity, determined by toxicity assay, were remarkably resis- tant to heating.

In the rat phrenic nerve-diaphragm preparation, the toxin caused a failure of neuromuscular transmission, after which subthreshold end- plate potentials could be detected. The frequency of spontaneous miniature potentials was altered only to a very slight extent. The toxin appeared to have no postsynaptic activity. Similarities between the properties of this neurotoxin and that described by Lee and Chang (1966) suggest that it is identical to p-bungarotoxin.

INTRODUCTION

Extensive studies of chemical synaptic transmission, especially a t the neuromuscular junction, have yielded a fairly detailed description of the sequence of steps involved in the secretion of neurotransmitters from nerve terminals, and of the subsequent interaction of neurotransmitters with postsynaptic receptors. The present level of our understanding of synaptic transmission has resulted almost entirely from anatomical and elec- trophysiological measurements. The application of biochemical tech- nology to obtain a molecular description of synaptic transmission has been difficult because synaptic structures usually constitute a tiny fraction of

Department of Anatomy, Washington University, St. LOU&, Missouri 63110.

Present address:

135 @ 1974 by John Wiley & Sons, Inc.

136 KELLY AND BROWN

the mass of tissue, and because no good assays have been available for macromolecules involved in synaptic function. Recently, however, one such assay did result from the observation by Lee, Tseng and Chin (1967) that a protein, a-bungarotoxin, isolated from the venom of the krait Bungurus multicinctus, bound specifically and irreversibly to a component of muscle with some of the characteristics of the acetylcholine receptor. This observation led to a large number of investigations which have verified that a-bungarotoxin and similar neurotoxins from cobras and sea-snakes do indeed bind to acetylcholine receptors (Sato, Abe and Tamiya, 1970; Miledi and Potter, 1971; Berg, Kelly, Sargent, Williamson and Hall, 1972). Subsequently, use has been made of a- bungarotoxin to estimate the number and distribution of acetylcholine receptors in neuromuscular preparations (Barnhard, Wieckowski and Chiu, 1971; Hartzell and Fambrough, 1972), and to purify the receptor from the electric eel (Fulpius, Cha, Klett and Reich, 1972).

Such progress in the study of acetylcholine receptors was possible because a-bungarotoxin, and the similar cobratoxins, can be easily purified, made radioactive with little loss of activity, and have a very high specificity and affinity for the acetylcholine receptor. By analogy, protein toxins which block neuromuscular transmission at sites other than the acetylcholine receptor, might prove equally useful in clarifying further the molecular basis of synaptic transmission. Botulinum toxin (Brooks, 1956), black widow spider venom (Longenecker, Hurlbut, Mauro and Clark, 1970), p-bungarotoxin (Lee and Chang, 1966) and notexin from the Australian snake Notechis scututus scututus (Karlsson, Eaker and Ryden, 1972) all seem to cause paralysis by a presynaptic block of transmitter release. In no case, however, has a complete examination of the physiological effects of these toxins been made, nor any receptor identified. In fact, it is unclear just what these receptors might be since so little is known of either the molecular mechanism of neurotransmitter secretion or its regulation.

The present work was begun in the belief that some of the more baf- fling properties of transmitter secretion from the presynaptic nerve terminals, such as the facilitation and depression of release during repetitive stimulation, might be elucidated by utilizing highly specific inhibitors of presynaptic function, which bind with sufficient strength to permit identification of their binding site. The venom of the snake, Bungurus multicinctus, seemed an ideal source of such inhibitors since it is readily available in large amounts, and contains presynaptic neurotoxins (Lee and Chang, 1966). Unfortunately, since no purification of these neurotoxins has been published it was necessary to evolve a purification procedure, verify the purity of the product, and give sufficient biochemical characterization to permit comparison between this neurotoxin and others. Physiological studies on the effect of this neurotoxin on neuromuscular preparations have shown considerable similarity to the observations of Lee and Chang (1966) on B-bungarotoxin. We tentatively conclude there-

A PRESYNAPTIC NEUROTOXIN 137

fore that the protein described below is identical with the p-bungarotoxin previously described.

METHODS

Materials. Crude venom from Bungarus multicinctus was obtained from Ross Allen Reptile Institute, Silver Springs, Florida, batch number 102171CCY.

Purification of toxin. 200 mg of crude venom was dissolved in 20 ml of 0.05M ammonium acetate and chromatographed on Sephadex CM50 (20 X 1.2 cm; cf. Bosman, 1972) using 500 ml of a linear gradient of 0.05M ammonium acetate, pH 5.0, to l M , pH 7.0. Peak I (Fig. 1) consisted mainly of a-bungarotoxin. Peak V (fractions 87 to 90), eluting at approximately 0.55M ammonium acetate, pH 6.5, had the highest activity in the toxicity assay (see below). Fractions were concentrated and desalted by ultrafiltration using an Amicon Diaflo filter (UM2, 43 mm). The protein was then lyophilized and stored at 0°C.

Isoelectric focusing. Isoelectric focusing on acrylamide gels followed the procedure of Wrigley (1968). Gels of dimensions 10 X 0.5 cm were prepared containing 7+% acrylamide, 0.2% bisacrylamide, lo/, LKB Ampholyne buffer, pH 3-10, 0.0657, N,N,N',N',-tetramethylethylene-diamine (TEMED), and 0.07%, ammonium persulfate. After layering 100 p1 of a 1 % Ampholyne, 5% sucrose solution on the gels, they were prerun at 1 mA/gel for 1 hr to remove the ammonium persulfate and estab- lish a pH gradient. A solution of protein in 10% sucrose was then layered on the gel and covered with a layer of 100 pl of 1% Ampholyne, 5% sucrose. The upper anodic reservoir contained 0.5% phosphoric acid, pH 2; the lower cathodic reservoir 0.01M NaOH, pH 12.5. The electrophoresis was performed in an apparatus from Hoefer

The flow rate was 14 ml/hr and 2.5 ml fractions were collected.

i -7.0 40

: 0 z 3 0

a + W V z

LL 0 m

$2 0

m a

IC

FRACTION NUMBER

Fig. 1. Chromatography of crude venom from Bungarus multicinctus on a column The absorption of each sample was measured, at 280 nm, of CM-Sephadex C-50.

after appropriate dilution.

138 KELLY AND BROWN

Scientific Instruments which was also used for sodium dodecyl sulfate (SDS'i-gel electrophoresis (see below). After 24 hr a t 400 volts, the final equilibrium positions of the proteins is reached, monitored by a visible band of cytochrome c . The gels were fixed with 10% trichloroacetic acid and the protein peaks identified by scanning a t 280 nm using a Gilford Model 2410 transport attachment, attached to a Brush pen recorder, Model 220.

Sedimentation equilibrium experiments were performed on a Spinco Model E ultracentrifuge with a split beam photoelectric scanning system (Lamers, Putney, Steinberg and Schachman, 1963), using the high speed tech- nique of Yphantis (1964). Solutions of approximately 0.3 absorbance units at 280 nm were used. All analyses were performed a t 20°C and 44,800 rpm using an AnF rotor. Direct scan readings of absorbance a t 280 nm were made after 22-26 hr a t the stated speed. The partial specific volume, U, was calculated from the amino acid composition.

All spectra were recorded on a Jasco Model J-10 Recording Spectropolarimeter, equipped for operation over the temperature range of 25 to 85 "C. Such temperature variation was accomplished by circulating water from an external heater through a jacket surrounding the cell. Temperatures of the sample cell were measured by a thermocouple. The cells (0.1 to 0.5 mm) were manufactured by the Pyrocell Company. No correction for the residual solvent content was made in the preparation of the protein solutions for circular dichroism studies, but for the sake of reproducibility, samples were kept under high vacuum for at least one day before use.

SDS-polyacrylamide gel electrophoresis. To analyze proteins by SDS gel electrophoresis, 50 p1 of samples of protein containing 10-50 pg of protein were heated in 1% SDS for 10 min at 100°C (nonreducing conditions) or in 1% SDS, 1% 2-mercaptoethanol for 10 min at 100°C (reducing conditions). An alternative reducing procedure, heating for 10 min a t 100°C in 1% SDS, 20 m M dithiothreitol, followed by the addition of iodoacetamide to a final concentration of 100 mM, was sometimes used, and produced results identical to those obtained by the former method. In all three methods, heating a t 100°C was followed by an overnight incubation a t 37°C. Poly- acrylamide gels were prepared and run according to the method of Davies and Stark (1970), except that, when reducing conditions had been used, the reservoirs contained 0.1 % mercaptoacetic acid. Staining, destaining, and scanning of gels have been described earlier (Berg e t al., 1972).

Amino acid analysis was performed on 1 mg of pure p- bungarotoxin using a Beckman Model 120B. Duplicate samples were hydrolyzed overnight in 6 N HCI (zn uacuo, 1lOOC). Tryptophan was estimated using the spectro- photometric method of Beaven and Holiday (1952). The average number of residues per 21,000 molecular weight chain was calculated using glycine as a unit residue, rounded off to the nearest whole number t o estimate molecular weight.

Tension measurements. Strips of rat diaphragm muscle approximately 1 cm wide, were anchored a t the rib end, and the tendon tied to a Grass FT .03 strain transducer. The output of the transducer was amplified using a Winston Electronics, Model SG 500 amplifier and recorded either on a Tektronix Model R 5030 or a Brush Model 220 recorder. The muscle was indirectly stimulated through the phrenic nerve using a suction electrode. Stimuli consisted of square pulses of 0.5 msec, 0.5 to 1.0 volt, delivered from a Grass S 44 stimulator, using an SIU 5 stimulus isolation unit. The Kreb's solution contained 134 m M NaCI, 4 m M KCI, 2 m M CaCI2, 1 m M MgS04, 1 m M KH,P04, 11 m M glucose, and 12 m M NaHCO? and was bubble dwith a 95% air, 5% C 0 2 mixture. Since continuous perfusion of the muscle bath with Kreb's solution containing the neurotoxin is extremely costly, the solution in the bath (5 ml) was replaced every 15 min in most experiments. For this reason muscles were usually exposed to toxin for only 15 min. All measurements were made a t room temperature, 22-25 'C.

Intracellular recordings were made with glass microelectrodes containing 3M KCl (10-40 Ma), using a microelectrode preamplifier

Sedimentation equilibrium.

The density a t 20°C was .99823 g/cm3.

Circular dichroism.

Amino acid analysis.

End-plate potential measurements.


Model 1090 from Winston Electronics Company. film or using a Grass kymograph camera, Model C4R. were as described above.

Recordings were made on Polaroid Stimulation and perfusion

RESULTS

Purity of neurotoxin

When the purified toxin was filtered through a Sephadex G-50 column (45 X 2 cm, 0.lM potassium phosphate, pH 7.5) more than 97% of the protein eluted in one peak with a relative elution volume (V,/V,) of 1.2. After isoelectric focusing in a polyacrylamide gel containing an ampholyte carrier of pH range 3-10, more than 90% of the protein was in a single band, 5.5 cm from the acidic end of a 10 cm gel. Finally, after heat denaturing the protein in 1% SDS, without reducing disulfide bonds, more than 94% of the protein was found in a single band (Fig. 2a). We therefore concluded that the toxin was free from major contaminants.

(a) SDS,no SH reducing agent

n RF

(b) SDS,+ SH reducing agent

1.0 0.8 0.6 0.4 0.2 RF

Fig. 2. Electrophoresis of p-bungarotoxin on SDS-polyacrylamide gels. (a) 10 pg of pure protein was heated in 1% SDS for 10 min at 100°C, then maintained at 37OC overnight. After adding Pyronin Y as a tracking dye, the sample was layered on the 10% acrylamide, 0.1% SDS gels. After about 5 hr a t 8 mA/gel, the gels were removed, stained with Coomassie blue, destained, and the peaks recorded by densitometry. The abscissae (RF) is the mobility relative to the tracking dye. The ordinate units are arbitrary. (b) The procedure was identical to that in (a) except 2 m M dithiothreitol was present during the denaturation, 10 m M iodoacetamide was added after the denaturation to alkylate the protein.

140 KELLY AND BROWN

Table 1 Amino Acid Composition of p-Bungarotoxin

Calculated Whole Number Values from Amino Acid Valuesa Values Leeb

Lysine Histidine Arginine Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Half-cystine Valine Methionine Isoleucine Leucine Tyrosine Phenylalanine Tryptophanc Total Residues

13 .3

15 .5 23.8 12 .o 6 . 1

12.7 8.06

18.2 11 .8 19.4 4 .4 2 . o 8 . 6 7 .2

13.2 6 .2 4 . 2

5.15 13 5

15 24 12 6

13 8

18 12 20

4 2 9 1

13 6 4

191 -

~

13 5

14 22 12

6 12 8

16 11 20 4 2 8 7

13 6 ?

179 -

a Calculated assuming a molecular weight of 21,000 daltons.

0 Estimated spectrophotometrically using the method of Beaven and Holiday (1952). From Lee (1972, p. 269)

-3 I I

49.2 49.4 49 6 49 8 50 0 50.2 2

[DISTANCE (cm) FROM A X I S OF ROTATION]

Fig. 3. tance from axis of rotation for a solution of p-bungarotoxin. the text.

Linearity of the logarithm of absorbance at 280 nm vs. the square of dis- Details are described in


Molecular weight and amino acid composition

By comparison of the relative elution volume of the toxin (V,/V, =

1.2) with that of chymotrypsinogen (1.15), cytochrome c (1.36) and C Y -

bungarotoxin (1.62) a molecular weight of the toxin of 21,000 * 2,000 was calculated. From the amino acid composition of the protein (Table l ) , the molecular weight is 21,800 daltons. In the absence of a published purification the quite close agreement between the amino acid composition given here and that quoted by Lee (1972) and reproduced in Table 1, is the major reason for assuming that our protein is identical to Lee's p-bungarotoxin, rather than some other highly toxic component of Bungarus multicinctus. From equilibrium sedimentation measurements, using the short-column method of Yphantis (1964), a weight average molecular weight of 18,500 f 400 daltons was calculated using a partial specific volume, U , calculated from the amino acid composition of 0.708 cc/g. is another indication of the homogeneity of the protein (Fig. 3).

The linearity of the relation between In (absorbance) and (radius)

Structure of toxin

The circular dichroism (CD) of a solution of the neurotoxin was determined a t 25°C in distilled water over the range of 190 to 260 nm. The CD spectrum showed negative extrema at 208 and 222 nm, a shape resem- bling the spectrum of synthetic polypeptides in an a-helical conformation (Greenfield and Fasman, 1969). From the rotational strength at 208 nm (-10,700 deg cm3 decimol-') and a t 222 nm (-9,400 deg cm2 decimol-') a helix content of approximately 23% could be calculated from the equations of Greenfield and Fasman (1969) and Chen and Yang (1971), which suggests that the toxin in solution contains an appreciable amount of random structure in addition to the helical component. The rotational strength at 222 nm decreased by approximately 10% when the toxin was heated to 85"C, indicating a marked stability in the structure of the toxin, an implication supported by the toxicity studies (see below).

The toxin appears to have 2 subunits held together by disulfide bonds. If the toxin was heated in 1% SDS a single band was observed in 10% polyacrylamide-SDS gels, (Fig. 2a) of RF = 0.50. However, if heating took place under reducing conditions (1% 2-mercapto ethanol or 20 mM dithiothreitol) two bands were found, RF = 0.710 =t .02 and 0.76 * .02. When compared with myoglobin (RF = 0.669), cytochrome c(RF =

0.720) and a-bungarotoxin (RF = 0.767), the twu bands for the reduced protein were calculated to have molecular weights of 8,800 =t 300 and 12,400 + 350, respectively. By densitometry of the stained gels, 41% of the total material was in the faster peak.

The two bands on SDS-gel electrophoresis under reducing conditions indicate that there are 2 subunits in the protein for the following rea- sons. Using the molecular weights of the two bands calculated from

142 KELLY AND BROWN

SDS-gel electrophoresis, and assuming one of each subunit per toxin molecule, 42% of the total protein should be in the faster band. From densitometry of the stained gels, the faster band constituted 41% of the total material, in very good agreement. The sum of their molecular weights, 21,200, is close to the values determined by gel filtration, amino acid composition, and equilibrium sedimentation. These data are all consistent with two chains of molecular weights approximately 8,800 and 12,400 daltons.

The possibility that the two bands were some fortuitous artifact of SDS gel electrophoresis, has been eliminated by partial separation of the two subunits by filtration on G-75 Sephadex under denaturing conditions, The toxin was labeled with 1 3 1 1 , using an identical procedure to that al- ready described for a-bungarotoxin (Berg et al., 1972). If the 1 3 1 1 -

B-bungarotoxin samples were denatured in SDS and dithiothreitol, and alkylated with iodoacetamide, chromatography on G-75 Sephadex (50 x 2 cm, 0.1M Tris pH 7.5, 1% SDS) did not clearly resolve the subunits. To determine whether any separation of subunits had occurred, fractions from the leading edge, trailing edge, and center of the peak region were mixed with 20 pg pure unlabeled p-bungarotoxin as carrier protein, denatured in SDS in the presence of dithiothreitol, and analyzed by SDS gel electrophoresis. The gels were stained with Coomassie blue to detect the two components of the carrier toxin. The regions of the gels containing these components could then be separated and counted in a Beck- man Scintillation counter using a toluene : triton scintillation fluid.

If separation of subunits had occurred on the column, samples from the leading edge of the radioactivity peak should have the least amount of the smaller 8,800 dalton component, and the trailing edge, the largest. This was found to be correct since, for samples of relative elution volume, 1.2, 1.33, and 1.48, the ratio of the radioactivity in the 8,800 dalton component to that in the larger component was found to be 0.50, 0.77 and 1.18, respectively, compared to a value of 0.69 for toxin which had not been fractionated on G-75 Sephadex. Since some separation of the components can be achieved in this way, the two bands on electrophoresis are not artifactual, but most probably represent two subunits of the toxin. Since dithiothreitol or 2-mercaptoethanol are required to separate the two subunits, they are presumably held together by disulfide bonds.

Toxicity The toxin we have characterized above is similar in amino acid com-

position to that described by Lee (1972) as a presynaptic inhibitor of neuromuscular transmission. To confirm that the protein whose purification is here described is indeed a presynaptic inhibitor and therefore most probably P-bungarotoxin some preliminary results are given of the biological properties of the purified material.

An inhibitor of neuromuscular transmission should be lethal, causing death by respiratory paralysis. To quantitate the toxicity of the purified


material mice were injected intraperitoneally with an appropriate dilution of toxin, and the time to die recorded. From a plot of the reciprocal of time to die against dose, a minimum lethal dose of 0.01 pg/g could be obtained. In agreement with earlier results (Chang and Lee, 1963), the minimum time to die (60-70 min) was reached with approximately five MLD units and was not decreased by further increase in dose. This long latency is also encountered in the response of the isolated neuromuscular junction to toxin (see below).

The toxin is remarkably stable to heating, as might be predicted from the number of half-cystines. For example, after heating a t 100°C for 10 min in physiological saline, 20% of the toxicity remained.

Action of the toxin on neuromuscular transmission

That the toxin, like p-bungarotoxin, does indeed interfere with neuromuscular transmission was confirmed using an isolated phrenic nerve-diaphragm preparation. In the first experiments we recorded the isometric tension produced by the muscle in response to electrical stimulation of the nerve. Both the twitch tension resulting from a single stimulus and the larger steady “tetanic” tension produced by repetitive stimulation were measured (Fig. 4a). Addition of toxin to the bath (10 mg/ml) at room temperature caused after a considerable, but reproducible, delay a decrease in the magnitude of both twitch and tetanic tensions, a distortion of the shape of the tetanic tension curve, and a marked reduction in twitch tension following a tetanus (Fig. 4b). Similar results were obtained by Chang and Lee using p-bungarotoxin (1963). Eventually after several hours, transmission was completely blocked, although the muscle responded to direct electrical stimulation with a twitch or tetanus identical to that recorded before toxin addition. Using extracellular electrode measurements, it was shown that conduction of action potentials in the phrenic nerve was not detectably affected either in velocity or amplitude by incubation in the toxin, under conditions

Tension measurements.

, , , I I I ‘ I * I

Fig. 4. Response of an isolated rat diaphragm-phrenic nerve preparation to single or repetitive stimulation. The stimulus record is reproduced below the tension measurements and consisted of several single stimuli and one train of stimuli a t 50/sec. Stimu- lus to the phrenic nerve was 0.7 volts for 0.5 msec.

144 KELLY A N D BROWN

Fig. 5. Time course of the action of p-bungarotoxin on twitch (-O-O-), tetanus (.A. .A.) tension, and tetanic attenuation (-0--0-). The response of a rat diaphragm-phrenic nerve preparation to 20 pg/ml p-bungarotoxin was determined by measuring the tension produced by single and repetitive indirect stimulation as in Fig. 3. The toxin was present for the first 15 niin of the experiment, after which un- bound toxin was removed by extensive washing. Initial twitch and tetanic tensions were 7 and 31 g, respectively.

which eliminated indirectly stimulated contraction. This does not, of course, exclude the possibility that the toxin blocks conduction in the fine terminals of the end-plate region.

Injection of the toxin into mice appeared to cause the same alterations in neuromuscular transmission as bath-applied toxin. When mice were injected with 20 MLD units of toxin and sacrificed a t different times after injection, strips of diaphragm muscle showed the same distorted tetanus tension curves and reduced posttetanic twitch tension, which became more pronounced with time, till transmission failed.

The observation that transmission failure eventually occurred in muscles removed from mice only 20 min after injecting the toxin, suggested that, as in the results of Lee and Chang (1966), the muscle need only be exposed for a brief period to reach complete intoxication. To verify

A P R E S Y N A P T I C NEUROTOXIN 145

this, a rat muscle was bathed in Kreb’s solution containing toxin (30 mg/ml) for 15 rnin a t room temperature, washed repeatedly, and the magnitudes of twitch and tetanic tension periodically measured. In addition, to quantitate the distortion in the shape of the tetanus tension, a parameter “tetanic attenuation” was defined as (Tmax-To.6)/Tmax, where T,,,, is the maximum tension produced by tetanic stimulation and To.5 is the tension 0.5 sec after the start of the stimulation period. The first observable indication of toxin action (Fig. 5) was an increase in tetanic attenuation which occurred after about 60 min, while twitch and tetanic tension both decreased rapidly in magnitude after a lag of about 110 min. By these parameters, the toxin affected neuromuscular transmission in an almost identical fashion whether or not i t was removed

400.

- c .- E - 300 -

1 I-

200 -

I I 10 20 30

DOSE (pg/ml)

Fig. 6. Time to fall to half the initial tension as a function of the concentration of The values were accumulated from

Twitch tension (-O--O-); tetanic tension (-A- -A-). the toxin in the bath, during a 15-min exposure. data such as those in Fig. 5.

from the bath after 15 rnin exposure. This implies that either the toxin was irreversibly bound to its site of action in the muscle, or that the neuromuscular junction was damaged during those 15 min in some way that is not fully expressed for several hours.

At a concentration of 30 pg/ml there is a considerable lag before the magnitude of the tension decreases (Fig. 5). To determine if this lag is dependent on dose, muscles were exposed to different concentrations of toxin for 15 rnin and the time measured for twitch and tetanus tensions to decrease to half their original values (Fig. 6). It is clear that tension did not decrease significantly faster when the toxin concentration was increased above 20 pg/ml. This result, reminiscent of the minimum time to die in the toxicity assays, indicates that the long time to fail is a consequence of the toxin’s mode of action, and is not a dose effect.

146 KELLY AND BROWN

Fig. 7. Subthreshold epp’s in toxin-treated muscle. Rat diaphragm was exposed to 14 rg/ml p-bungarotoxin for 15 min. Records were made for 10 sec a t 10 stimuli/sec. A sample of three consecutive end-plate potentials, 105 min after addition of toxin, is illustrated. The vertical bar represents 2 mV, and stimuli, indicated by arrows, were 100 msec apart.

I 50 100 150 200 250 300

TIME ( m i d

Fig. 8. Frequency of miniature end-plate potentials after toxin treatment. 20 pg/ml of a-bungarotoxin was present in the bath during the first 15 min of the experiments. The results represent the average of six experiments, the bars representing the standard error of the mean during each measurement period. In 3 out of 86 measurements frequencies of greater than 15/sec were observed, but since these were also observed occasionally in untreated muscles, they were excluded from the sample.

End-plate potential measurements. Except for small differences in the absolute values of the dose-response curve, which could well reflect small difficulties in experimental procedure, the failure in neuromuscular transmission caused by the toxin closely paralleled that caused by p- bungarotoxin. It remained to be demonstrated that the toxin did indeed act presyna ptically to block release. Intracellular recording from end- plate regions of muscle fibers showed action potentials a t early times after toxin addition; at later times, subthreshold end-plate potentials were observed (Fig. 7) as was reported by Lee and Chang (1966) for P-bungarotoxin treated fibers. The presence of mepp’s of normal amplitude in such fibers indicated that the toxin had negligible action postsynaptically


but was acting to reduce the quanta1 content of the evoked release. In addition, on many occasions the end-plate potentials were followed by a very striking “delayed release” of mepp’s (Fig. 7) similar to that described for example by Rahamimoff and Yaari (1973) in frog muscle. A more detailed description of this phenomenon is in preparation.

In the absence of stimulation the presence of the toxin caused an initial three- to fourfold increase in the frequency of mepp’s (Fig. 8), followed by a steady decrease. While in agreement with the results of Lee and Chang (1966) using 6-bungarotoxin this contrasts markedly with the results obtained with black widow spider venom where a several hundred- fold increase in mepp frequency was observed (Longenecker e t al. 1970). This difference makes it unlikely Lhat the mechanism proposed for transmission failure by black widow spider venom, namely depletion of available quanta by rapid spontaneous release, applies to the action of p- bungaro toxin.

DISCUSSION

This paper describes the purification of a protein from the venom of Bungarus multicinctus with a molecular weight of 22,000 and two unequal subunits. The amino acid sequence closely resembles that given by Lee (1972) for the neurotoxin, p-bungarotoxin, although he quotes a much larger molecular weight of 28,500. Despite this difference we prefer to believe that the toxin described here is p-bungarotoxin and have so named it in order not to confuse the literature with two names for the same protein. Clearly the question will not be unequivocally resolved until Chang and Lee publish the purification and characterization of their neurotoxin.

A comparison of the structure of p-bungarotoxin with that of the only other snake venom to act presynaptically, notexin, shows that they differ markedly in structure; the former with a molecular weight approximately 22,000 daltons and two subunits, the latter a single polypeptide chain of 13,600 (Karlsson e t al., 1972). Some homology may exist, however, between the larger subunit of p-bungarotoxin and notexin. Since it is of bacterial origin, botulinum toxin would not be expected to resemble the snake neurotoxins and with a molecular weight of 167,000 (Beers and Reich, 1969), does not. No purification of the active component or components of black widow spider venom has yet been reported.

In addition to the similarity in amino acid composition, the other basis for identifying this neurotoxin with p-bungarotoxin, is the similarity in biological properties. The toxicity, the time to die, and the time for failure of neuromuscular transmission as a function of dose for p-bungarotoxin (Lee and Chang, 1966) closely resemble the data given in this paper. The larger doses needed in this case could well reflect the lower temperature of incubation, the lower frequency of stimulation, and the

148 KELLY AND BROWN

fact that, in our studies, the toxin was only present in the bath for the first 15 min of the experiment. This fact, that only a brief exposure to toxin is necessary, as well as the early onset of “tetanic attenuation,” are likewise properties shared by this toxin and p-bungarotoxin. Finally, the production of subthreshold epp’s and the small temporary elevation in mepp frequency caused by both preparations, lead to the conclusion that the toxins are, if not identical, then very closely related.

There are a few points of difference between the two investigations. Briefly, these include a marked calcium requirement for the increase in mepp frequency; some indications from non-Poisson failures that conduction block may play a role in failure in some fibers; a large enhance- ment of delayed release; and, from our unpublished electron microscopic studies, accumulation of “coated vesicles’’ (Heuser and Reese, 1973) in toxin- treated terminals, rather than opened synaptic vesicles (Chen and Lee, 1970). These apparent differences are being investigated further.

The physiological consequences of the addition of toxin include a small increase followed by a decrease in mepp frequency, a reduction in quanta1 content, and enhanced delayed release. The morphological consequences (Marshall and Kelly, unpublished observations; Chen and Lee, 1970) of the presence of toxin are a small depletion of vesicles, and markedly distended mitochondria. From these observations, a model can be con- structed, which, although by no means unique, fits the data quite well. In this model the toxin binds to the nerve terminal and causes either directly or indirectly an accumulation of calcium inside the terminal from extracellular sources. Mitochondria, which are known to act as scavengers of calcium inside cells, act in a manner analogous to the sarco- plasmic reticulum in muscle to reduce the calcium levels within the cytoplasm of the terminal. However, as the internal calcium level in the mitochondria increases, oxidative phosphorylation is diminished (Lehninger, 1970), and the production of ATP falls. Thus failure of transmission arises from energy starvation. While this model has soime appealing features, considerably more data must be obtained either to prove or disprove it. Hopefully such data will also lead to a better understanding of the metabolism of the nerve terminal, especially of the very important mechanisms for the elimination of calcium after evoked release of transmitter.

One might hope that the specificity of binding of the toxin to the presynaptic terminal would reflect the presence there of some physiologically active receptor whose function is not yet appreciated. So far the data have offered no suggestions as to what such a receptor might be, although experiments using radioactively labeled toxin might help provide an answer to this question, if specific binding can be shown. If the physiological role of the toxin can be established and the nature and distribution of its binding sites shown using radioactively labeled toxin, a useful molecular probe of the presynaptic terminal would be available.

What can be said of the mechanism of action of this toxin?


The authors are indebted to Ms Nan Burgess for excellent technical assistance, to Mr. Stephen Oberg for performing the equilibrium density sedimentation measurements, and to Dr. Elizabeth Simons for the amino acid analysis. This work was supported by USPHS grant number NS 09878-2.

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Biochemical and physiological properties of a purified snake venom neurotoxin which acts presynaptically