Binding of Plg and Plm to HK. (A) Surface plasmon resonance. Human HK was immobilized on CM5 sensor chips, and binding affinities for Plgs or Plms (25-400 nM) were measured by a single-cycle assay at 25°C. Binding curves for Plg-Glu³¹¹ and Plm-Glu³¹¹ are shown in red and for Plg-Lys³¹¹ and Plm-Lys³¹¹ in blue. Data were fitted with a 1:1 Langmuir binding model (dashed line). Association rate constants (k_a), dissociation rate constants (k_d), and equilibrium dissociation constants (K_D) are as listed in the table. (B) Coprecipitation. Human recombinant HK (2 μg) was incubated for 30 minutes in 500 μL of buffer with or without 2 μg of active site–inhibited Plm-Lys³¹¹ or Plm-Glu³¹¹. HK was precipitated with anti-HA IgG bound to magnetic beads. Proteins were eluted with SDS nonreducing sample buffer and size fractionated on a 10% polyacrylamide gel, followed by staining with Coomassie Blue (left). Similar to the left panel, except controls with Plm-Lys³¹¹ or Plm-Glu³¹¹ but no HK were included (center). Similar to the left panel, except human HK was replaced with mouse HK (right). For all panels, positions of molecular mass standards (kDa) are shown to the left of each image, and positions of controls for HK and Plms are shown to the right of each image. Human and mouse HK normally contain 2 bands. The 2 arrows for the Plm controls are needed because human and mouse Plms migrate slightly differently. (C) Coprecipitation experiment for human recombinant LK run in an identical manner to the studies for HK in panel B. Note that coprecipitated Plms migrate above LK, whereas they run lower than HK, on SDS–polyacrylamide gel electrophoresis.