Purification of the human placental α2‐macroglobulin receptor

Abstract

The α₂‐macroglobulin receptor was solubilized from human placental membranes, purified and characterized. Affinity cross‐linking of labelled ligand to intact membranes showed a receptor size compatible with 400–500 kDa. The membranes were solubilized in 3‐[(3‐cholamidopropyl)dimethylammonio]propane sulfonate (CHAPS) and affinity chromatography was performed using Sepharose‐immobilized α₂‐macroglobulin‐methylamine with elution in buffer containing 2 mM EDTA, pH 6.0. SDS‐PAGE of the resulting receptor preparation showed a predominant approx. 440 kDa band (reducing conditions) and some minor accompanying proteins of 70–90 kDa and 40 kDa. The yield was 400–800,μg receptor preparation per placenta. The receptor preparation immobilized on nitrocellulose bound the α₂‐macroglobulin‐trypsin complex with a dissociation constant of about 400 pM. ¹²⁵I‐iodinated receptor preparation bound almost quantitatively to Sepharose‐immobilized α₂‐macroglobulin‐methylamine in the presence of CHAPS alone, and bound 70–80% in the presence of 0.2% SDS. The labelled proteins were separated in the presence of 0.2% SDS by gel filtration or SDS‐PAGE (unboiled samples). The 440 kDa protein accounted for the major part of the binding, although some approx. 80 kDa proteins, perhaps proteolytic degradation products, also showed binding activity.