Affiliations: Laboratoire de Biochimie Cellulaire et Moléculaire en Nutrition, EP CHRS 616 | Laboratoire de Biochimie Médicale I, Faculté de Médecine, Université H. Poincaré, Nancy, France | Instituto di Industrie Agrarie della Universita’ degli Studi, via S. Michele, 4‐56124 Pisa, Italy | LEIMA, National Institute for Research in Agronomy (INRA), Nantes Centre, Protein Group, Nantes, France
Abstract: The aim of the present work was to study the binding of [{}^{125}I]‐BLGA (\beta ‐lactoglobulin variant A) to the plasma membrane fraction of hybrid cells. This binding increased as a function of time with on‐rate and off‐rate constant at 4.47 \pm 0.18\times 10^{6} M^{-1 } min^{-1 } and 0.17 \pm 0.07\,min^{-1}, respectively (n = 3). The saturation study showed a single binding site type corresponding to a K_{d } at 8.26 \pm 2.98 nM and 14.02 \pm 2.61\times 10^{12} sites per mg of the plasma membrane protein (n = 3). Competitive of binding BLGA was observed with BLGA, complexed with retinol and also with RBP (retinol‐binding protein). Gel filtration of [{}^{125}I]‐BLGA incubated with Triton X‐100 solubilized membrane showed the formation of a ligand‐receptor complex. Cross‐linking of the tracer to plasma membrane showed a complex with a M_{r} at 69 kDa, suggesting a receptor M_r of 51 kDa, as seen by autoradiography of SDS‐PAGE.
