Characterization and scatchard binding analysis of adrenal digitalis-like material

Culture medium conditioned by the adrenocortical tumor cell line Y-1 has been fractionated by HPLC to determine whether the ouabain-like binding activity (OLB) of this medium co-purifies with digoxin immunoreactivity. Furthermore, we have evaluated whether this medium contains the ability to inhibit the ion translocating action of the sodium pump and how this material purifies on HPLC. These activities are not homogenous, however, maximal activity in each assay was found in the same HPLC fraction. The OLB of this fraction was not reduced by alkaline hydrolysis or trypsinization, heating in air at 150 degrees C did reduce activity. Scatchard binding analysis of this fraction indicated the presence of a single class of specific competitive inhibitor of the binding of radiolabelled ouabain to human erythrocytes. The inhibitor showed a Hill coefficient of unity.