Purification of the cardiac 1,4-dihydropyridine receptor using immunoaffinity chromatography with a monoclonal antibody against the α2δ subunit of the skeletal muscle dihydropyridine receptor

Abstract

The 1,4-dihydropyridine receptor associated with L-type Ca 2+ channels was purified about 1700-fold from porcine cardiac sarcolemmal membranes using a simple and rapid (ca. 8 h) two-step procedure: wheat germ agglutinin affinity chromatography followed by immunoaffinity chromatography with a monoclonal antibody (MCC-1) against the α 2 δ subunit of the skeletal muscle Ca 2+ channel with a glycine elution buffer (pH 3). Gel electrophoresis of this purified sample under non-reducing conditions revealed a major polypeptide band with molecular weight of 190 kDa, which was separated under reducing conditions to a 155 kDa band and 2–3 bands with M r about 20 kDa, corresponding to α 2 and δ subunits, respectively. The peptide band corresponding to the α 1 subunit was not detected in this gel electrophoresis. However, the α 1 subunit without bound α 2 δ was selectively eluted from MCC-1 Sepharose with 1% Triton X-100. A 190 kDa band corresponding to the α 1 subunit was visualized by fluorography and by silver staining in the fraction eluted with Triton X-100. Electrophoretically, the amount of α 1 was smaller than that of the α 2 subunit in the purified sample obtained here.