Valdivia H H, Martin B M, Ramírez A N, Fletcher P L, Possani L D
Departamento de Bioquímica, Universidad Nacional Autónoma de México, Cuernavaca.
J Biochem. 1994 Dec;116(6):1383-91. doi: 10.1093/oxfordjournals.jbchem.a124691.
Four novel Na+ channel-blocking toxins (numbered 6 to 9) were purified from the venom of the scorpion Centruroides noxius Hoffmann by gel filtration and high-performance liquid chromatography and their chemical and pharmacological properties were characterized. Amino acid analysis and SDS-PAGE of the pure toxins showed them to be composed of approximately 65 amino acid residues with a molecular mass of approximately 7,500 Da. The amino acid sequences of the newly isolated toxins displayed substantial similarity to those of previously isolated and characterized C. noxius toxins. Toxin 7, the most active toxin from this venom, selectively blocked the whole-cell inward Na+ current (INa) from guinea pig ventricular myocytes without altering the K+ or Ca2+ currents. Using rat brain synaptosomes, the specific binding parameters of 125I-Toxin 7 were determined: KD = 40 pM and Bmax = 1.8 pmol/mg protein. The binding was independent of membrane potential and was displaced by all Na+ channel-toxins from C. noxius venom, with K0.5s ranging from 60 pM to 60 nM. Tityus gamma-toxin, a toxin representative of beta-scorpion toxins, totally displaced 125I-Toxin 7 binding, but AaH II and Lqq V toxins, representative of alpha-scorpion toxins, had no effect. All four C. noxius toxins inhibited [3H]GABA uptake by synaptosomes with IC50 s similar to KD s. The toxin effect was not synergistic with veratridine. From these results, it was concluded that the newly purified toxins exert their effects by binding to Site 4 of the voltage-sensitive Na+ channel and must, therefore, be classified as beta-scorpion toxins.
通过凝胶过滤和高效液相色谱从墨西哥毒蝎（Centruroides noxius Hoffmann）毒液中纯化出四种新型钠通道阻断毒素（编号为6至9），并对其化学和药理特性进行了表征。对纯毒素进行氨基酸分析和SDS-PAGE分析表明，它们由大约65个氨基酸残基组成，分子量约为7500 Da。新分离毒素的氨基酸序列与先前分离和表征的墨西哥毒蝎毒素具有显著相似性。毒素7是这种毒液中活性最强的毒素，它选择性地阻断豚鼠心室肌细胞的全细胞内向钠电流（INa），而不改变钾电流或钙电流。利用大鼠脑突触体，测定了125I-毒素7的特异性结合参数：KD = 40 pM，Bmax = 1.8 pmol/mg蛋白质。这种结合与膜电位无关，可被墨西哥毒蝎毒液中的所有钠通道毒素取代，K0.5值范围为60 pM至60 nM。β-蝎毒素的代表毒素——Tityusγ-毒素完全取代了125I-毒素7的结合，但α-蝎毒素的代表毒素——AaH II和Lqq V毒素则没有影响。所有四种墨西哥毒蝎毒素均抑制突触体对[3H]GABA的摄取，IC50值与KD值相似。毒素的作用与藜芦碱无协同作用。从这些结果可以得出结论，新纯化的毒素通过与电压敏感性钠通道的位点4结合发挥作用，因此必须归类为β-蝎毒素。
