Organization and phylogenetic analysis of kappa-bungarotoxin genes from Bungarus multicinctus (Taiwan banded krait).

Two genomic DNAs were isolated from the liver of Bungarus multicinctus (Taiwan banded krait) encoded kappa1-bungarotoxin and kappa3-bungarotoxin precursors, respectively. They shared virtually identical overall organization with three exons separated by two introns and a high degree of nucleotide-sequence identity with alpha-neurotoxin genes, including similar intron insertions. This suggests that kappa-neurotoxins and alpha-neurotoxins might have originated from a common ancestor. The consensus transcriptional factor binding sites within the promoter regions of these genes indicate that their transcriptions are, at least in part, regulated under the same mechanism. Comparative analyses on kappa-bungarotoxin and alpha-neurotoxin genes revealed that the protein-coding regions of exons were much more diversified than introns except for the signal peptide domain. Phylogenetic analyses on the exon and intron regions of kappa-bungarotoxin and alpha-neurotoxin genes showed that the evolution of exon regions were not in consensus with that of intron regions. The ratio of nonsynonymous to synonymous substitution is higher than 1, reflecting the occurrence of an adaptive selection during the evolution of kappa-bungarotoxins. In contrast to a conserved size of the second intron, segmental insertions and/or deletions within the first intron accelerate the evolutionary divergence of kappa- and alpha-neurotoxin genes.

Nucleus basalis lesions decrease alpha- and kappa-bungarotoxins binding in rat cortex.

A unilateral ibotenic acid lesion of the nucleus basalis magnocellularis in the rat, which is known to produce a reduction in cortical choline acetyltransferase activity and acetylcholine release, produces a decrease of 125I-alpha-bungarotoxin and 125I-kappa-bungarotoxin binding sites in the frontoparietal cortex of the lesioned hemisphere. This decrease can be observed at two weeks following the lesion and persists for up to twelve weeks. The results suggest that a population of bungarotoxin binding sites may have a presynaptic localization.

Amino acid sequences of three beta-bungarotoxins (beta 3-, beta 4-, and beta 5- bungarotoxins) from Bungarus multicinctus venom. Amino acid substitutions in the A chains.

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.