Novel neurotoxins from Bungarus multicinctus: Purification, Characterization, and Gene Organization.

• Main Authors: Chung Ca-Ling, 鍾家齡
• Other Authors: L. S. Chang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/92885586686262424366

碩士 === 國立中山大學 === 生物醫學科學研究所 === 89 === Three three-finger proteins (BM8, BM11, BM14) were isolated from Bungarus multicinctus (Taiwan banded krait) venom using successive chromatography on a ion-exchanger column and reverse phase HPLC column. BM8 and BM14 contain 82 amino acid residues including 10 cysteine residues, and BM11 contain 68 amino acid residues including 10 cysteine residues. Unlike snake venom cardiotoxins and a-neurotoxins, the three proteins contain two additional cysteine residues in their N-terminal region. Noticeably, only two amino acid substitutions occurred at positions 37 and 38 were observed between BM8 and BM14. CD measurement revealed that their secondary structures were dominant with b-sheet structure as noted with cardiotoxins and a-neurotoxins. However, the gross conformations of BM8, BM11 and BM14 were not the same as evidenced by CD spectra and acrylamide quenching studies. In contract to BM8, BM11 and BM14 exhibit an activity on blocking [3H]QNB binding to muscarinic acetylcholine receptor (mAChR). Modification of Lys residues in BM14 resulted in a decreased binding activity, indicating that the Lys residues may be involved in the mAChR binding. The genomic DNA with the size of approximate 2.3 kb and 2.4 kb, respectively, encoded the precursors of BM14 and BM11 was amplified by PCR. The genes shared virtually identical overall organization with snake venom cardiotoxin andα-neurotoxin genes, composed of three exons and two introns. Comparison of BM11 and BM14 genes with cardiotoxins and a-neurotoxins genes showed that the exon regions were more diversified than intron regions. This implicated that the accelerated evolution may be involved in the evolution of these genes in order to acquire newly arising functions. Nevertheless, several transcriptional factor binding sites including SP-1, CACCC-binding factor and NF-KB were highly conserved in the promoter regions. It reflected a common regulation mechanism in controlling the transcription of these genes. These observations indicate that the three proteins are evolutionarily related to cardiotoxins and a-neurotoxins.