Smith C, Phillips M, Miller C
J Biol Chem. 1986 Nov 5;261(31):14607-13.
Charybdotoxin is a high-affinity specific inhibitor of the high-conductance Ca2+-activated K+ channel found in the plasma membranes of many vertebrate cell types. Using Ca2+-activated K+ channels reconstituted into planar lipid bilayer membranes as an assay, we have purified the toxin from the venom of the scorpion Leiurus quinquestriatus by a two-step procedure involving chromatofocusing on SP-Sephadex, followed by reversed-phase high-performance liquid chromatography. Charybdotoxin is shown to be a highly basic protein with a mass of 10 kDa. Under our standard assay conditions, the purified toxin inhibits the Ca2+-activated K+ channel with an apparent dissociation constant of 3.5 nM. The protein is unusually stable, with inhibitory potency being insensitive to boiling or exposure to organic solvents. The toxin's activity is sensitive to chymotrypsin treatment and to acylation of lysine groups. The protein may be radioiodinated without loss of activity.
大毒蜥毒素是一种高亲和力的特异性抑制剂，可作用于许多脊椎动物细胞类型质膜中发现的高电导钙激活钾通道。我们以重组到平面脂质双分子层膜中的钙激活钾通道作为分析对象，通过两步法从蝎子黄肥尾蝎的毒液中纯化了该毒素。第一步是在SP-葡聚糖凝胶上进行色谱聚焦，然后进行反相高效液相色谱。结果表明，大毒蜥毒素是一种质量为10 kDa的高碱性蛋白质。在我们的标准分析条件下，纯化后的毒素抑制钙激活钾通道的表观解离常数为3.5 nM。该蛋白质异常稳定，其抑制效力对煮沸或暴露于有机溶剂均不敏感。毒素的活性对胰凝乳蛋白酶处理和赖氨酸基团的酰化敏感。该蛋白质可以进行放射性碘化而不丧失活性。
