Evidence for the existence of hyaluronectin-binding proteins in the plasma membranes

Abstract

This report documents for the first time the existence of specific binding proteins for hyaluronic acid binding protein (hyaluronectin) in the plasma membranes of normal and transformed cells. Firstly, we showed the specific binding of hyaluronic acid binding protein to the cell surface of normal rat heart fibroblasts (NRHF) by saturation and competition methods using ¹²⁵I-labeled hyaluronic acid binding protein and calculated the binding dissociation constant (0.43 × 10^-13 M). In order to identify hyaluronectin-binding protein on the cell surface, plasma membranes isolated from rat brain, liver and flbrosarcoma were separated by SDS-PAGE and transferred to nitrocellulose paper by electroblotting. Incubation of the transferred membrane proteins with ¹²⁵I-labeled hyaluronectin in the presence of non-ionic as well as ionic detergents revealed two prominent bands of approximate molecular mass of 37 kDa and 40 kDa in brain, liver and flbrosarcoma. The specificity of the binding [¹²⁵I]hyaluronectin to 37-kDa and 40-kDa membrane proteins was further confirmed, as the intensity of the bands was reduced in the presence of a 20-fold excess of unlabeled hyaluronectin. We discuss our observations on hyaluronectin-binding membrane proteins in the context of hyaluronectin-mediated cellular functions.