Binding of asialotransferrins by purified rat liver plasma membranes

Interaction of four different asialotransferrins (human types 1, 2, and 3, and rabbit asialotransferrin) with purified plasma membranes from the rat liver was studied by using a direct binding assay. Binding of rabbit asialotransferrin, possessing a single biantenary glycan, was too weak to establish a complete binding curve, but the human asialotransferrins, possessing two glycan attachments, did yield binding data over a sufficiently wide range of concentrations for Scatchard plot analysis. At 22 degrees C, a quantity of plasma membrane equivalent to 1 mg of membrane protein bound comparable quantities (12.3-12.8 pmol) of the asialotransferrin types with an association constant of 1.5 X 10(6) M-1 for type 1, 1.4 X 10(7) M-1 for type 2, and 1.1 X 10(8) M-1 for type 3. At 4 degrees C, the number of binding sites and the association constants were reduced, more so for asialotransferrin types 2 and 3 than for type 1. At both temperatures, the shapes of the Scatchard plots for all three asialotransferrin types were similar: in the low range of bound asialoprotein (below 0.6-0.7 nM), each plot exhibited two to three convex peaks, tentatively identified as restricted domains of positive cooperativity; in the higher range, however, the plots were linear. The findings are consistent with the view that the binding sites involved in the binding of asialotransferrin are homogenous.