Figure 8.
Binding of lysozyme to megalin. In A, megalin immobilized on a BIA sensor chip was incubated with the indicated concentrations of lysozyme. From the kinetic parameters, a Kd value of 0.32 μmol/L was calculated (see under Materials and Methods). In B, the receptor was preincubated with 5 μmol/L RAP resulting in 1800 response units. No further increase in response was achieved by subsequent addition of 0.5 μmol/L lysozyme (or RAP) indicating complete inhibition of ligand binding by RAP. As a control, 0.5 μmol/L lysozyme were added to the receptor chip without prior addition of RAP (200 response units). In C, binding of 2 μmol/L lysozyme to native megalin was tested in the presence or absence of EDTA, or to megalin reduced by 0.5% dithiothreitol, 6 mol/L guanidine hydrochloride. Adding EDTA or reducing the receptor changed the Kd to 39 μmol/L (as compared to 0.32 μmol/L for functional megalin). Similar results were obtained with DBP, RBP, and β2-M (not shown). The arrows indicate time points of ligand addition to the sensor chip.