PURIFICATION OF THE CARDIAC 1,4-DIHYDROPYRIDINE RECEPTOR USING IMMUNOAFFINITY CHROMATOGRAPHY WITH A MONOCLONAL-ANTIBODY AGAINST THE ALPHA(2)DELTA SUBUNIT OF THE SKELETAL-MUSCLE DIHYDROPYRIDINE RECEPTOR

The 1,4-dihydropyridine receptor associated with L-type Ca2+ 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 alpha2delta subunit of the skeletal muscle Ca2+ 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 alpha2, and delta subunits, respectively. The peptide band corresponding to the alpha1 subunit was not detected in this gel electrophoresis. However, the alpha1 subunit without bound alpha2delta was selectively eluted from MCC-1 Sepharose with 1% Triton X-100. A 190 kDa band corresponding to the alpha1 subunit was visualized by fluorography and by silver staining in the fraction eluted with Triton X-100. Electrophoretically, the amount of alpha1 was smaller than that of the alpha2 subunit in the purified sample obtained here.