Dekleva M L, DasGupta B R, Sathyamoorthy V
Food Research Institute, University of Wisconsin, Madison 53706.
Arch Biochem Biophys. 1989 Oct;274(1):235-40. doi: 10.1016/0003-9861(89)90435-9.
Botulinum neurotoxin (NT) has two distinct structural regions called L and H chains (approximately 50 and approximately 100 kDa, respectively). Although the H chain is responsible for binding of the NT to neuronal cells, it is not known which of the subunits is internalized and therefore responsible for causing the blockage of acetylcholine release in susceptible neuronal cells. In this report we describe for the first time the preparation of type A NT which is selectively radiolabeled at either the L or the H chain subunit. Such NT preparations will be useful as tools for determining the distribution of L and H chains in poisoned neuronal cells and the role that each subunit plays in inducing toxicity. The L and H chains of the NT (approximately 150 kDa) were separated, purified, and then individually radiolabeled by reductive methylation of the lysine residues using [3H]- or [14C]formaldehyde. The labeled L and H chains were reconjugated with the complementary unlabeled L and H chains. Formation of -S-S- and noncovalent bonds between the L and H chains regenerated the approximately 150 kDa NT. Autoradiographs of sodium dodecyl sulfate polyacrylamide gels confirmed that each reconstituted NT preparation was labeled at only one subunit chain. NT selectively labeled at either the L or the H chain had specific radioactivities of ca. 25-30 and 45-55 microCi/mumol, respectively, and toxicity (mouse LD50/mg protein) values of 2.2 +/- 1.1 X 10(7) and 3.0 +/- 1.0 X 10(7), respectively. A linear increase in the specific radioactivity of L and H chain subunits was observed with increasing concentrations of 3H- or 14C-labeled formaldehyde in the reaction mixture and with increasing concentrations of L or H chain in the reaction mixture.
肉毒杆菌神经毒素（NT）有两个不同的结构区域，称为轻链（L链）和重链（H链）（分别约为50 kDa和100 kDa）。尽管H链负责NT与神经元细胞的结合，但尚不清楚哪个亚基会被内化，进而导致易感神经元细胞中乙酰胆碱释放受阻。在本报告中，我们首次描述了分别在L链或H链亚基上进行选择性放射性标记的A型NT的制备方法。此类NT制剂将有助于确定中毒神经元细胞中L链和H链的分布，以及每个亚基在诱导毒性中所起的作用。将NT（约150 kDa）的L链和H链分离、纯化，然后使用[³H]-或[¹⁴C]甲醛对赖氨酸残基进行还原甲基化，分别对其进行放射性标记。将标记的L链和H链与互补的未标记L链和H链重新结合。L链和H链之间形成的-S-S-键和非共价键使约150 kDa的NT得以再生。十二烷基硫酸钠聚丙烯酰胺凝胶的放射自显影片证实，每种重组NT制剂仅在一个亚基链上被标记。分别在L链或H链上进行选择性标记的NT的比放射性分别约为25 - 30和45 - 55 μCi/μmol，毒性（小鼠LD50/毫克蛋白）值分别为2.2±1.1×10⁷和3.0±1.0×10⁷。随着反应混合物中³H-或¹⁴C-标记甲醛浓度的增加以及反应混合物中L链或H链浓度的增加，观察到L链和H链亚基的比放射性呈线性增加。
