Blockade by botulinum neurotoxin B of catecholamine release from adrenochromaffin cells correlates with its cleavage of synaptobrevin and a homologue present on the granules.
Botulinum neurotoxin type B blocks transmitter release via a selective endoproteolysis of the small clear vesicle membrane protein synaptobrevin that is essential for neuro-exocytosis. In view of the distinct characteristics of exocytosis of adrenochromaffin granules and considering 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-purified antibodies against a synaptobrevin synthetic peptide were used to quantify its concentrations in subcellular fractions of bovine adrenal medulla. The latter, as well as density gradient centrifugation and size-exclusion chromatography, showed that > 70% of the protein copurifies with the granules and their marker, dopamine beta-hydroxylase. Notably, much lower concentrations of synaptobrevin and synaptophysin were found in chromaffin granules than in synaptic small clear vesicles (approximately 9% and approximately 2%, respectively); however, isolated granule membranes exhibited greater enrichments (approximately 35% and approximately 9%). A second immunoreactive protein was colocalized with synaptobrevin 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 correlation between the extent of botulinum neurotoxin B-induced inhibition of secretion and their selective proteolysis following introduction of the toxin into intact chromaffin cells. On the basis of these collective findings, it is concluded that these proteins occur on chromaffin granules and one or both are essential for exocytosis.
Foran
P
P
Wolfson Laboratories, Department of Biochemistry, Imperial College, London, U.K.
Lawrence
G
G
Dolly
J O
JO
D000302:Adrenal Cortex / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D001905:Botulinum Toxins / Q000494:pharmacology*; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002458:Cell Fractionation; D002540:Cerebral Cortex / Q000378:metabolism*; D002837:Chromaffin Granules / Q000187:drug effects / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D050683:R-SNARE Proteins; D013347:Subcellular Fractions / Q000378:metabolism; D016708:Synaptophysin / Q000302:isolation & purification / Q000378:metabolism
D000906:Antibodies; D002395:Catecholamines; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D016708:Synaptophysin; D001905:Botulinum Toxins