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Fig. 1. Ca2+ induces the formation of multiple MgATP-dissociatable
complexes in situ. Chromaffin cells were permeabilised with 20 µM digitonin
in KGEP and maintained in the absence or presence of 20 µM Ca2+,
with or without the inclusion of 2 mM MgATP. After 15 minutes, aliquots were
removed and assayed for catecholamines. Cells in some wells were solubilised
in Triton X-100 and used to estimate the total amount of catecholamine
remaining and the amounts released (A) were expressed (±s.d.
n=4) as a percentage of the total cell content, calculated by summing
the amounts released with the level remaining. The remaining cells were lysed
in KGEP, 2 mM PMSF was added and membrane-enriched fractions were isolated, as
detailed in the Materials and Methods. The latter were dissolved in 50 mM
Tris-HCl, pH 5.8 containing 1% SDS before adding 4x sample buffer and
being subjected to SDS-PAGE. The separated proteins were either transferred
directly to PVDF (B) or extracted from the gel by boiling in fresh sample
buffer and re-electrophoresed before transfer (C). The PVDF membranes were
blotted with antibodies raised against recombinant SNAP-25. Immunodetection of
primary antibody binding was performed as described in Materials and Methods.
Note that the left and right panels show signals for two different bands
(Mr=25 K and 83 K, respectively).
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