Isolated light chains of botulinum neurotoxins inhibit exocytosis. Studies in digitonin-permeabilized chromaffin cells.
The effects of botulinum neurotoxins or their light and heavy chain subunits were investigated in digitonin-permeabilized adrenal chromaffin cells. Because these cells are permeable to proteins, the toxin had direct access to the cell interior. Botulinum type A neurotoxin and its light chain subunit inhibited Ca2+-dependent catecholamine secretion in a dose-dependent manner. The heavy chain subunit had no effect. Inhibition required introduction of the neurotoxin or light chain into the cell and was not seen when intact cells were incubated with these proteins. The inhibition of secretion by type A neurotoxin and light chain was incomplete, the maximal response being 65%. The inhibition was not overcome by increasing Ca2+ concentrations. The action of the light chain was irreversible and rapid. Botulinum type E neurotoxin also inhibited secretion in a dose-dependent manner. Its potency was increased 30-fold following mild trypsinization, which nicked the single chain protein to the dichain form. In contrast to the results seen with types A and E, botulinum type B neurotoxin did not inhibit secretion, while its light chain totally abolished secretion. Trypsinization of the neurotoxin produced the dichain form, which did not inhibit secretion. Reduction of the trypsinized neurotoxin with dithiothreitol produced inhibition equivalent to that seen with the purified light chain subunit. Isolated type A heavy chain had no effect on the inhibitory action of type A or B light chains. The data demonstrate that the ability of botulinum neurotoxins to inhibit secretion is confined to the light chain region of these proteins. Furthermore, while the botulinum neurotoxin types A, B, and E have similar macrostructures, they are not identical with respect to their biological activities.
Bittner
M A
MA
Department of Pharmacology, University of Michigan Medical School, Ann Arbor 48109.
DasGupta
B R
BR
Holz
R W
RW
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001905:Botulinum Toxins / Q000302:isolation & purification / Q000494:pharmacology*; D002417:Cattle; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004072:Digitonin; D005089:Exocytosis / Q000187:drug effects*; D007700:Kinetics; D046911:Macromolecular Substances; D009498:Neurotoxins; D014357:Trypsin
D046911:Macromolecular Substances; D009498:Neurotoxins; D014357:Trypsin; D001905:Botulinum Toxins; D004072:Digitonin