Characterization of the binding and uptake of MPO by cultured endothelial cells. (a) Characterization of cell MPO association. BAECs were incubated with increasing concentrations of MPO (2–13 nM) for 2 hours at 37°C and harvested by scraping (total) or after exposure to trypsin (trypsin-resistant). Cell-associated MPO activity was about 10% of total MPO activity added (not shown). (b) The influence of temperature on cellular MPO binding and transport. BAECs exposed to MPO (13 nM) at 37°C and 4°C were harvested at different time points and MPO activity was determined. ^†P < 0.05 for MPO activity in total cell lysates at 37°C versus 4°C; *P < 0.05 for MPO activity in trypsin-resistant compartments at 37°C versus 4°C. (c) The influence of glycosaminoglycans on cell MPO binding. BAECs were pretreated with chondroitin sulfate (Chondr, 150 μg·ml^–1), heparin (Hep, 150 μg·ml^–1), or the low–molecular weight heparin enoxaparin (Enox, 150 μg·ml^–1) for 45 minutes, washed with HBSS, and then exposed to MPO (13 nM) at 37°C. After washing again, cell-associated MPO was assessed by enzyme activity analysis and cellular MPO protein content by immunoblotting. “MPO hc ” denotes the immunoreactivity of the heavy chain (59 kDa) of MPO. (d) The effect of endoglycosidases on MPO binding. BAECs were pretreated with heparitinase, heparinase, and chondroitinase (all 8 mU·ml^–1) for 2 hours at 37°C, washed, and exposed to MPO (13 nM) at 4°C for 2 hours. Cell-associated MPO enzyme activity was then determined. *P < 0.05 for MPO alone versus pretreatment with heparin and enoxaparin (c) and versus heparitinase and heparinase pretreatment (d). (e) Binding competition analysis of MPO and xanthine oxidase (XO). BAECs were incubated with MPO (1 μg·ml^–1) and increasing concentrations of XO (0–100 μg·ml^–1, equivalent to 0–100 mU·ml^–1) for 2 hours at 37°C. Cells were harvested as above and MPO enzyme activity determined. Values represent mean ± SD.