Fig. 2. Effects of exogenous proteases on Ca²⁺-triggered fusion and CV membrane proteins. (A) Isolated free-floating CVs in suspension were treated with the indicated protease concentrations. Following protease neutralization and washing, recovered CVs were suspended in B-IM buffer. A fraction of each sample was taken for protein extraction and analysis, and the remainder was assayed for Ca²⁺-triggered fusion. Control CVs (black circle) were sampled and tested immediately after preparation (0 hours; n=16); incubated controls (open circle) were left for 1 hour at 25°C in the absence of protease (n=26). CVs were incubated with 700 (purple triangle; n=5), 3500 (inverted purple triangle; n=9) and 35,000 (purple diamond; n=4) units/ml trypsin, respectively, with 2000 units/ml of papain (blue hexagon; n=9), or with 100 units/ml clostripain (yellow square; n=3) for 1 hour at 25°C. Data presented are means±s.e.m. of the normalized data from a total of 32 separate CV preparations made over the course of two seasons. (B) Isolated CVs, treated as in (A), but with a 4 hour incubation time; control CVs (0 hours) are represented by a solid black curve as in (A). Control samples without protease (x; n=5) were incubated in parallel with samples containing 2000 units/ml papain (blue hexagon; n=6) or 100 units/ml clostripain (yellow square; n=3). (C) Differential effects of exogenous proteases on the profile of hydrophobic proteins extracted from CVs. CVs treated as described in (B) were analyzed by SDS–PAGE and silver staining. Incubations (4 hour) were with buffer only (lane 1), 100 units/ml clostripain (lane 2) or 2000 units/ml papain, as indicated. 1 hour incubations yielded similar protein profiles, although changes were less pronounced (not shown). (D) Endogenously docked CV–PM preparations were treated (1 hour) with the indicated protease concentrations, neutralized, washed, suspended in B-IM buffer and assayed for Ca²⁺-triggered fusion. Controls (black circle) were sampled and tested 1 hour after preparation, and other samples were incubated (1 hour, 25°C) with 700 (purple triangle), 14,000 (inverted purple triangle) and 35,000 (purple diamond; n=4) units/ml trypsin, respectively, with 3000 units/ml of papain (blue hexagon), or with 100 units/ml clostripain (yellow square). Data are representative examples of one to three determinations.

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