Premium
Purification of the human placental α 2 ‐macroglobulin receptor
Author(s) -
Jensen P.H.,
Moestrup S.K.,
Gliemann J.
Publication year - 1989
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(89)81105-6
Subject(s) - chaps , chemistry , methylamine , chromatography , sepharose , size exclusion chromatography , macroglobulin , affinity chromatography , trypsin , receptor , membrane , alpha 2 macroglobulin , gel electrophoresis , nitrocellulose , ligand (biochemistry) , biochemistry , enzyme
The α 2 ‐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 α 2 ‐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 α 2 ‐macroglobulin‐trypsin complex with a dissociation constant of about 400 pM. 125 I‐iodinated receptor preparation bound almost quantitatively to Sepharose‐immobilized α 2 ‐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.