Fig. 3. Average effects of exogenous proteases on fusion and CV SNARE proteins. Isolated CVs were treated with proteases and their hydrophobic proteins extracted, separated by SDS–PAGE and the constituent SNARE proteins analyzed by quantitative immunoblotting. An aliquot of each sample was also analyzed for fusion. The SNARE complements of isolated CVs are shown in the bar graph following 1-4 hour incubations at 25°C with 2000 units/ml papain (n=10-14 for the individual SNAREs), 100 units/ml clostripain (n=8), or 700 and 3500 units/ml (n=5 each) trypsin, as indicated. Papain treatment reduced VAMP, SNAP-25 and syntaxin to, on average, 341±108, 62±21 and 4±1 copies/CV, respectively. Clostripain reduced VAMP, SNAP-25 and syntaxin to 424±143, 93±17 and 20±5, respectively. Also shown is the extent of post-treatment CV–CV fusion elicited by a maximal [Ca²⁺]_free (1 mM).