Partial purification of the rat erythrocyte ceruloplasmin receptor monitored by an electrophoresis mobility shift assay.
Ceruloplasmin (Cp), the main copper transport glycoprotein found in the blood, delivers its copper to intracellular proteins via a plasma membrane receptor protein. Electrophoresis mobility shift assays (EMSAs) were originally developed to detect DNA-protein or RNA-protein binding. A new EMSA involving protein-protein binding has been developed in order to follow the extraction and purification of the rat erythrocyte Cp receptor. The assay utilized rat Cp (rCp) with a radiolabel (125I-rCp), native polyacrylamide gel electrophoresis (PAGE), and detergent-solubilized erythrocyte plasma membrane. Five detergents, at concentrations of 1.0%, were readily screened for their ability to extract the receptor using this assay. Triton X-100 and sodium dodecyl sulfate (SDS) showed the greatest yields of rCP-receptor complex with large mobility shifts to higher molecular weight. Apo-rCp as well as native rCp bound to the receptor. Maximum rCp-receptor complex formation was observed after a 5-min incubation at 25 degrees C with dimerization or higher order aggregation occurring by 10 min. The binding of rCp to the receptor was specific as unlabeled rCp showed displacement of 125I-rCp from the 125I-rCp-receptor complex, while unlabeled bovine serum albumin did not. The Triton X-100-extracted receptor had an M(r) of 150,000 as determined by Sephadex G-200 chromatography. The SDS-extracted receptor maintained activity after 80 degrees C for 10 min, so SDS-PAGE was also used to determine the M(r) of the receptor. With this technique the subunit M(r) of the receptor was approximately 56,000.(ABSTRACT TRUNCATED AT 250 WORDS)
Stern
R V
RV
Chemistry Department B-164, Florida State University, Tallahassee 32306-3006.
Frieden
E
E
D000818:Animals; D002570:Ceruloplasmin / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004910:Erythrocyte Membrane / Q000378:metabolism*; D008970:Molecular Weight; D011485:Protein Binding; D051381:Rats; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000302:isolation & purification*; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; D012995:Solubility
D003432:Cross-Linking Reagents; D003902:Detergents; D011956:Receptors, Cell Surface; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; C040519:ceruloplasmin receptor; D002570:Ceruloplasmin