Abstract. a-Bungarotoxin, a polypeptide of mol wt 8000 purified from the venom of Bungarus multicinctus, blocks irreversibly and specificallythe excitation by cholinergic agonists on the isolated electroplax and on purified membrane fragments in vitro. The toxin also blocks the in vitro binding of decamethonium to a protein recently isolated from electric tissue. This observation strengthens our earlier conclusion that this protein is the cholinergic receptor macromolecule.At a glance, the most evident and simple manner to characterize and identify the physiological receptor of acetylcholine in an excitable membrane is to use compounds that are structurally analogous to acetylcholine and thus present a high affinity for the cholinergic receptor site. However, it is now well established that in most excitable membranes there exist several-distinct classes of sites, all of which are able to bind cholinergic ligands. Among them are, in addition to the physiological receptor site, the catalytic and allosteric sites of the enzyme acetylcholinesterase (AcChE).l The use of cholinergic ligands thus meets with a difficult problem of specificity.2Interestingly enough, in the course of the past few years it has been shown that certain toxins from snake venoms, although completely unrelated structurally to acetylcholine, nevertheless act much like curare. One of them is a-bungarotoxin (a-Bgt), a basic polypeptide of mol wt 8000, which has been extensively studied by Lee and his associates.3-5 a-Bgt is purified from the venom of an elapid snake from Taiwan (Bungarus multicinctus) and the purified toxin gives irreversible neuromuscular blocking effects. In addition, d-tubocurarine protects against the action of a-Bgt. From these findings Lee and Chang4 have concluded that a-Bgt combines irreversibly with the cholinergic receptor at the motor endplate.We report here some experiments carried out with purified a-Bgt and various preparations derived from the electric organ of Electrophorus electricus. It is shown that in vivo, with the isolated electroplax, a-Bgt irreversibly blocks the depolarization caused by bath application of carbamylcholine, a cholinergic agonist. The same effect is observed with purified electric-organ membrane fragments using the in vitro assay of Kasai and Changeux.6 In addition, in agreement with the early observation of Lee and associates, d-tubocurarine protects against a-Bgt blockade. Finally we tested a-Bgt on the binding of 1241

The two dissimilar composite polypeptide chains (A and B) in beta1-bungarotoxin were isolated as their reduced and carboxymethylated derivatives as reported in the preceding paper. The N-terminal sequences were determined with a sequenator up to the 39th residue for the RCM-A chain and up to the 25th residue for the RCM-B chain with repetitive yields of 90-95%. The tryptic and chymotryptic peptides from the two chains were isolated and their structures were determined by manual Edman degradation together with dansyl-Edman and carboxypeptidases A and Y. To complete the primary structures of the two chains, information on the tryptic peptides derived from the maleylated derivatives of the two chains was also used. The completed amino acid sequence of the A chain containing 120 residues (molecular weight, 13,500) is similar to that of notexin, a presynaptic neurotoxin from Australian tiger snake venom, and phospholipases A from other snake venoms. The amino acid sequence of the 60 residues in the B chain (molecular weight, 7,000) bears no resemblance to any basic polypeptides from snake venoms. The B chain probably plays a significant role by interacting with some components in presynaptic membranes of neurosmuscular junctions.

From the venom of an Elapidae snake from Southeast Asia, Bungarus multicinctus, a neurotoxic polypeptide, designated a-bungarotoxin, has been isolated by column chromatography on CM-Sephadex C-25 or C-50 followed by rechromatography on CM-Sephadex or cellulose. Homogeneity was proved by polyacrylamide gel electrophoresis, analytical ultracentrifugation, amino acid analysis and end group analysis. The molecular weight was estimated giving a formula weight of 7983. The toxin consists of a single polypeptide chain of 74 amino acid residues crosslinked by five disulfide bridges. The amino acid sequence has been determined by Edman degradation of the reduced and 5-aminoethylated toxin and of its two cyanogen bromide fragments and by analyzing chymotryptic, tryptic and thermolytic peptides of these cyanogen bromide fragments. Tryptic peptides of the whole reduced, 5-aminoethylated toxin gave further information and confirmed the primary structure. Comparing the amino acid sequence with those of homologous neurotoxins of cobra and sea snake venoms close similarities are observed especially between Naja nwea -toxin and -bungarotoxin indicating 50% sequence homology.

The two most basic beta-bungarotoxins (beta 3- and beta 4-toxins) and another, less neurotoxic beta-bungarotoxin (beta 5-toxin) were purified from Bungarus multicinctus venom, by a combination of CM-Sephadex C-25 column chromatography and Sephadex G-75 gel filtration. The three toxins consisted of two dissimilar polypeptides (A chain, 120 amino acid residues; B chain, 60 residues). The LD50 values of the beta 3- and beta 4-toxins were 0.066 micrograms and 0.072 micrograms/g of mouse, respectively, and their phospholipase A activities were 43.2 and 36.5 units/mg of toxin, respectively. beta 5-Toxin was weaker in neurotoxicity (LD50, 0.13 micrograms/g of mouse) than the others, and its phospholipase activity was 47.6 units/mg of toxin. Each toxin was separated into RCM-A and RCM-B chains after reduction and S-carboxymethylation. The RCM-polypeptides were maleylated and digested with TPCK-trypsin. The tryptic peptides were sequenced with manual Edman degradation or the dansyl-Edman method. The final alignment of the tryptic peptides from the respective RCM-polypeptides was deduced on the basis of the amino acid sequences of the A and B chains of beta 1-bungarotoxin (beta 1-toxin). The amino acid sequences of the A chains of the beta 3- and beta 4-toxins were identical but differed from those of the A chains of the beta 1- and beta 2-toxins by 4 amino acid substitutions in the COOH-terminal portions (residues 109-120) and substitution at position 87. The amino acid sequences of the B chains of the beta 3- and beta 4-toxins differed from each other, but they were identical with those of the B chains of the beta 1- and beta 2-toxins, respectively. The amino acid sequence of the A chain of beta 5-toxin differed from that of the A chain of beta 1-toxin by consecutive substitutions in residues 55-60 and substitutions at positions 23, 87, and 89. The amino acid sequence of the B chain of beta 5-toxin was identical with those of the B chains of beta 1- and beta 3-toxin. From our results on the effects of the amino acid displacements found in the A chains on the neurotoxicity, it was concluded that the COOH-terminal portion in the A chains was not essential to their neurotoxicity, whereas the region of residues 55-60 in the A chains appeared to participate in the constitution of the neurotoxically active site of the beta-toxins.

beta 2-Bungarotoxin (beta 2-toxin) was isolated from the venom of Bungarus multicinctus by means of CM-Sephadex C-25 column chromatography, Sephadex G-75 gel filtration and CM-Sephadex C-25 column rechromatography. beta 2-Toxin consisted of two dissimilar polypeptides, a (120 amino acid residues) and B (60 amino acid residues) chains, crosslinked by an interchain disulfide bond. The neurotoxicity (LD50) and phospholipase activity of beta 2-toxin were 0.029 micrograms/g of mouse and 48.9 units/mg of toxin, respectively, and both the activities were slightly weaker than those (0.019 micrograms/g and 60.9 units/mg) of beta 1-bungarotoxin (beta 1-toxin). beta 2-Toxin was reduced and carboxymethylated and then its RCM-A and -B chains were separated. Each RCM-chain was maleylated and then digested with TPCK-trypsin. The tryptic peptides were sequences by manual Edman degradation or the dansyl-Edman method, and the total alignment of the tryptic peptides from each RCM-chain was deduced based on the amino acid sequences of the A and B chains of beta 1-toxin. The amino acid sequence of the B chain of beta 2-toxin differed from that of the B chain of beta 1-toxin by 22 amino acid substitutions, while those of their A chains were identical. We concluded that the variation in the amino acid sequence of the B chains did not significantly affect the neurotoxicity of the beta-toxins. The amino acid sequences of the B chains of the two beta-toxins were homologous to those of proteinase inhibitors from snake venoms and mammalian pancreas, but no inhibitory activity of the two beta-toxins on proteinases was observed.

The crude venom of Formosan cobra (Naja naja atra) was separated into 12 fractions by CM‐Sephadex (C‐50) chromatography with ammonium acetate buffer by two‐stage gradient elution. Fr. I was proved to be a mixture of nucleotides and nucleosides. The toxicity was found in Fr. VIII (neurotoxic), X (cardiotoxic), XI (cardiotoxic), and XII (cardiotoxic). Glycerophosphatase, alkaline phosphomonoesterase, and 5′‐nucleotidase were found in Fr. III, V and IX, respectively. Phosphodiesterase was distributed in Frs. VI and VII, while lecithinase‐A (phospholipase‐A) was found in Frs. IV and V. Frs. VIII, IX and XII seem to be homogenous by the n‐terminal analysis and the paper electrophoresis.

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A weakly neurotoxic component (Ls-III) was isolated by CM-cellulose column chromatography from the venom of a sea snake Laticauda semifasciata. The content of component LsIII was about 10-20% of the venom as determined by u.v. absorption at 280nm. Component LsIII was homogeneous on rechromatography and disc electrophoresis, and its molecular weight was shown to be 7100 by ultracentrifugation and 7300 by sodium dodecyl sulphate-polyacrylamide-gel disc electrophoresis. The isoelectric point of component LsIII was pH7.2. Component LsIII consisted of 66 amino acid residues including 10 half-cystine residues. The LD(50) of component LsIII by intramuscular injection was 1.24mug/g body wt. for mice and 0.45mug/g for baby chicks, which is about eight to ten times less toxic than erabutoxins a, b and c, all of which are contained in the same venom. Experiments with three isolated muscle preparations from different species indicated that component LsIII was a post-synaptically acting toxin, the action of which was easily reversed by washing.