Botulinum neurotoxin type A radiolabeled at either the light or the heavy chain.
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.
Dekleva
M L
ML
Food Research Institute, University of Wisconsin, Madison 53706.
DasGupta
B R
BR
Sathyamoorthy
V
V
D001905:Botulinum Toxins; D002250:Carbon Radioisotopes; D004591:Electrophoresis, Polyacrylamide Gel; D005557:Formaldehyde; D007202:Indicators and Reagents; D007553:Isotope Labeling / Q000379:methods; D046911:Macromolecular Substances; D009498:Neurotoxins; D014316:Tritium
D002250:Carbon Radioisotopes; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D009498:Neurotoxins; D014316:Tritium; D005557:Formaldehyde; D001905:Botulinum Toxins