HK cleavage by PKa and PK. Western blots of time courses of cleavage of HK (200 nM) by (A) PKa (2 nM) or (B) recombinant PK species (200 nM) in PBS at 37°C. In panel B, “vehicle” indicates reactions without PK. Some reactions in panel B were run in the presence of 750 nM C1INH or 750 nM CTI. At indicated time points, samples were removed into reducing sample buffer, size fractionated by SDS-PAGE, and analyzed by western blotting using polyclonal anti-HK IgG. Positions of standards for uncleaved HK (HK) and heavy and light chains of cleaved HK (HKa) are indicated at the right of each image. (C) Varying concentrations of HK were incubated with 1 nM α-kallikrein (left panel) or 200 nM PK-R371A (right panel) in PBS at 37°C. After 2 hours of incubation, reactions were stopped by ice-cold ethanol and BK concentration was determined by ELISA. Each point represents a single measurement. (D) Kinetic parameters for cleavage of HK by PKa or PK-R371A determined from the curves in panel C.