Prekallikrein structure over time after interaction with CHO-K1 cells.

<p>H-kininogen (100 nM) was incubated for 30 min with CHO-K1, and after removing H-kininogen by aspiration, prekallikrein (100 nM) was added for 15 min, 30 min, 1 h or 2 h at 37°C. Alternatively, prekallikrein was added to cells at different time points in the absence of assembled H-kininogen. At the end of each time point, the incubation buffer was collected, and both the incubation buffer and the cell samples were treated with reducing sample buffer. Immunoblotting was performed with anti-plasma kallikrein U691.10 for detecting plasma kallikrein fragments containing the sequence C₃₆₄TTKTSTR₃₇₁. (A) Cells with H-kininogen: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg); (7) H-kininogen (0.7 μg). (B) Cells without H-kininogen: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg). (C) Incubation buffer with H-kininogen: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg); (7) H-kininogen (0.7 μg). (D) Incubation buffer without H-kininogen: (1) 15 min; (2) 30 min; (3) 1 h; (4) 2 h; (5) plasma kallikrein (0.9 μg); (6) prekallikrein (1.6 μg)