Specific binding of [ 3H]heparin to bovine granulosa cell membranes

Abstract

Bovine granulosa cell membranes from small (SFM) and large (LFM) antral follicles were incubated with [ 3H]heparin, a commercial radioactively labeled glycosaminoglycan (GAG). Binding was specific, reversible, saturable, and dependent on time, pH, ionic strength and divalent cations. SFM exhibited different [ 3H]heparin binding characteristics compared to LFM. The addition of a physiological concentration of calcium (2 mM) yielded significant differences ( P < 0.02) in [ 3H]heparin binding for SFM (87590 ± 4206 dpm/10 6 cells) compared to LFM (55230 ± 2816 dpm/10 6 cells). SFM and LFM showed maximum [ 3H]heparin binding at pH 6.5 and pH 5.5, respectively. Increasing the ionic strength by addition of 0.07-2.0 M NaCl interfered with binding. Addition of unlabeled heparin (0.1–100 μg/ml) displaced [ 3H]heparin bound to SFM and LFM in a dose-dependent manner, as did dextran sulfate, a non-GAG sulfated branched polysaccharide. Commercial chondroitin sulfate ABC displaced the bound [ 3H]heparin only at doses between 50 and 500 mg/ml. GAGs purified from FF suppressed binding 39% at a concentration of 5.9 mg/ml. Photomicrographs of fluorescein-labeled heparin bound to granulosa cells showed localized areas of heparin binding to the cell surface. These experiments demonstrated that the GAG heparin specifically bound to bovine granulosa cell membranes, and that significant differences existed between the binding characteristics of SFM and LFM.