BLOCKADE BY BOTULINUM NEUROTOXIN-B OF CATECHOLAMINE RELEASE FROM ADRENOCHROMAFFIN CELLS CORRELATES WITH ITS CLEAVAGE OF SYNAPTOBREVIN AND AHOMOLOG PRESENT ON THE GRANULES

Botulinum neurotoxin type B blocks transmitter release via a selective endoproteolysis of the small clear vesicle membrane protein synaptobr evin that is essential for neuro-exocytosis. In view of the distinct c haracteristics of exocytosis of adrenochromaffin granules and consider ing the controversy over the presence of synaptobrevin on the latter, this study aimed to determine the molecular basis of the inhibition by this toxin of secretion from chromaffin cells. Thus, affinity-purifie d antibodies against a synaptobrevin synthetic peptide were used to qu antify its concentrations in subcellular fractions of bovine adrenal m edulla. The latter, as well as density gradient centrifugation and siz e-exclusion chromatography, showed that >70% of the protein copurifies with the granules and their marker, dopamine beta-hydroxylase. Notabl y, much lower concentrations of synaptobrevin and synaptophysin were f ound in chromaffin granules than in synaptic small clear vesicles (sim ilar to 9% and similar to 2%, respectively); however, isolated granule membranes exhibited greater enrichments (similar to 35% and similar t o 9%). A second immunoreactive protein was colocalized with synaptobre vin on chromaffin granules; in view of its susceptibility to the toxin and lower M(r), it is assumed to be cellubrevin and, also, because of its high homology. Involvement of synaptobrevin and cellubrevin in Ca 2+-triggered granule exocytosis was established by the demonstrated co rrelation between the extent of botulinum neurotoxin B-induced inhibit ion of secretion and their selective proteolysis following introductio n of the toxin into intact chromaffin cells. On the basis of these col lective findings, it is concluded that these proteins occur on chromaf fin granules and one or both are essential for exocytosis.