Figure 2.

Binding of [¹²⁵I]LDL by CD36-transfected cells after modification by MPO-generated nitrating intermediates. (a) [¹²⁵I]LDL was incubated with isolated human MPO (30 nM), glucose (100 μg/mL), glucose oxidase (20 ng/mL), and NO₂^– (0.5 mM) in sodium phosphate buffer supplemented with DTPA (100 μM) for 8 hours at 37°C (complete system, NO₂-LDL) as described in Methods. Reactions were stopped by addition of BHT (40 μM) and catalase (300 nM), and then lipoproteins (5 μg/mL) were incubated with CD36- or vector-transfected 293 cells at 4°C for 3 hours in the appropriate media containing additional catalase (300 nM) and BHT (20 μM). LDL modified by dialysis against copper for 5 hours (Cu²⁺ oxLDL, 5 μg/mL) was used as a positive control. Cellular binding of lipoproteins was subsequently determined as described in Methods. ^AP < 0.001 for comparison versus LDL modified in the presence of MPO- and a H₂O₂-generating system (MPO/GGOx/LDL). (b and inset) [¹²⁵I]LDL (0.2 mg/mL) was incubated with isolated human MPO (30 nM), glucose (100 μM), glucose oxidase (20 nM), and the indicated concentrations of NO₂^– in sodium phosphate buffer supplemented with DTPA (100 μM) and NaCl (100 mM) overnight at 37°C. Cellular binding of lipoproteins (10 μg/mL) was subsequently determined as described in Methods. Data represent the mean ± SD of triplicate determinations of a representative experiment performed 3 times.