Direct binding of 125I-RAP to LRP fragments E4-C10 (A) and C5-C7 (B). 125I-RAP was added to receptor fragments (250 ng E3-C10 or 100 ng C5-C7) coated in 96-well microtiter plates as described in Materials and Methods. Triplicate experiments were used to calculate a mean and SD for each data point. In each experiment the total amount of 125I-RAP added ranged from 0.5 nmol/L to 200 nmol/L and the concentration of free125I-RAP was calculated by counting the well buffer after overnight binding. The amount of 125I-RAP bound was determined by counting the dry wells after washing. Nonspecific binding was determined by subtracting the value of wells in which125I-RAP was bound without calcium and in the presence of 40 mmol/L EDTA. The Kd values were determined to be 2.13 ± 0.67 for the fragment E3-C10 (A) and 1.60 ± 0.09 for C5-C7 (B). Inset to each panel is the Scatchard plot of the Langmuir isotherm calculated from the data.
