Reductive chain separation of botulinum A toxin — a prerequisite to its inhibitory action on exocytosis in chromaffin cells

Abstract

Cleavage of the disulfide bond linking the heavy and the light chains of tetanus toxin is necessary for its inhibitory action on exocytotic release of catecholamines from permeabilized chromaffin cells [(1989) FEBS Lett. 242, 245-248; J. Neurochem., in press]. The related botulinum A toxin also consists of a heavy and a light chain linked by a disulfide bond. The actions of both neurotoxins on exocytosis were presently compared using streptolysin 0-permeabilized bovine adrenal chromaffin cells. Botulinum A toxin inhibited Ca*+-stimulated catecholamine release from these cells. Addition of dithiothreitol lowered the effective doses to values below 5 nM. Under the same conditions, the effective doses of tetanus toxin were decreased by a factor of five. This indicates that the interchain S-S bond of botulinum A toxin must also be split before the neurotoxin can exert its effect on exocytosis.

Adrenal chromaffin cells contain functionally different SNAP‐25 monomers and SNAP‐25/syntaxin heterodimers

“…Interestingly the molecular weight of the SNAP-25 immunoreactive band in the blots was about 60 kDa, that is about 35 kDa larger than that of authentic SNAP-25. We harvested the chromaffin vesicle fractions (11)(12)(13)(14) from the sucrose gradients, isolated their membranes (see Section 2), and analyzed them further. Again, in unboiled samples the anti-SNAP-25 antibody labelled a band of 60 kDa (Fig.…”

“…Absorbance at 280 nm after precipitation of the protein with 10% (w/v) TCA was measured to conveniently locate chromaffin vesicles in the gradient while arylsulfatase served to identify fractions containing lysosomes and other subcellular membranes [26]. The fractions enriched in chromaffin vesicles (fractions [11][12][13][14] were recovered from the gradient, diluted 1:4 with 20 mM MOPS (pH 7.0), 5 mM EDTA and centrifuged for 1 h at 100 000 x g. The purified chromaffin vesicles were osmotically lysed by addition of a 10-fold excess of 20 mM MOPS (pH 7.0) with 5 mM EDTA. Chromaffin vesicle membranes were recovered by centrifugation for 1 b at 100000xg and washed twice in potassium glutamate medium (see above) containing 6 mM magnesium acetate.…”

“…Catecholamine release from adrenal chromaffin cells, vasopressin release from neurohypophysial terminals and insulin release from pancreatic 13 cells is inhibited by botulinum neurotoxin A [14][15][16][17][18]. However, the variable cleavage of SNAP-25 in pancreatic [3 cells, the incomplete inhibition of vasopressin and catecholamine release and the preferential attack of ATP-dependent priming by botulinum neurotoxin A suggested that this toxin may have molecular targets of different sensitivities [14][15][16][17][18][19][20][21].…”

Release of vasopressin from isolated permeabilized neurosecretory nerve terminals is blocked by the light chain of botulinum A toxin

“…21 In these neuroendocrine cells reductive conditions enhance the inhibitory effect on catecholamine secretion of the two-chain form of botulinum A toxin. 20 Furthermore, the light chain alone is fully active. 5 .…”

Botulinum neurotoxin light chains inhibit both Ca2+‐induced and GTP analogue‐induced catecholamine release from permeabilised adrenal chromaffin cells

“…Chromaffin cells express all three neurotoxin substrates [17,18] which can be isolated in a complex [18] and Ca2+-dependent exocytosis in these cells is sensitive to neurotoxins that target VAMP (tetanus and botulinum B neurotoxins) [19][20][21][22][23][24] or SNAP-25 (botulinum A and E neurotoxins) [23,[25][26][27][28]. Insulin-secreting cells are also sensitive to these two classes of neurotoxins [29][30][31] and studies on these cells have suggested that VAMP is essential for Ca2+-induced secretion but not for insulin release activated by the non-hydrolysable GTP analogue, GTPTS, since this was insensitive to tetanus toxin and botulinum B neurotoxin [29].…”