Calcium-dependent release of norepinephrine from permeabilized PC12 cells is inhibited by approximately 48 and approximately 112 kDa fragments of botulinum neurotoxin type E.
Permeabilized PC12 cells exhibit a Ca(2+)-stimulated norepinephrine secretory pathway which is sensitive to botulinum neurotoxin serotypes A, B and E [Lomneth R., Martin T.F.J. and DasGupta B. R. (1991) J. Neurochem. 57: 1413-1421]. Two novel amino terminal fragments of the 150 kDa neurotoxin serotype E (approximately 112 and 48 kDa), produced by digestion with pepsin, were tested in permeabilized PC12 cells. The intracellular inhibitory activity of the approximately 112 kDa amino terminal fragment, like that of the 150 kDa neurotoxin, was progressively enhanced after trypsinization and dithiothreitol reduction. The approximately 50 kDa C-terminal half of the heavy chain therefore does not contribute to the enhancement of inhibitory activity. The approximately 48 kDa amino terminal light chain-like fragment completely inhibited release of norepinephrine, with an IC50 = 500 pM (more potent than the light chain isolated after digestion with trypsin) not requiring reduction with dithiothreitol. These results clarify the molecular basis of activation of neurotoxin by trypsin and dithiothreitol.
Lomneth
R
R
Department of Food Microbiology and Toxicology, University of Wisconsin, Madison 53706.
Gimenez
J
J
Martin
T F
TF
DasGupta
B R
BR
D000818:Animals; D001905:Botulinum Toxins / Q000494:pharmacology*; D002118:Calcium / Q000502:physiology*; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004229:Dithiothreitol; D006868:Hydrolysis; D009638:Norepinephrine / Q000378:metabolism*; D016716:PC12 Cells; D010446:Peptide Fragments / Q000494:pharmacology*; D014357:Trypsin
D010446:Peptide Fragments; D014357:Trypsin; D001905:Botulinum Toxins; D002118:Calcium; D004229:Dithiothreitol; D009638:Norepinephrine