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 alpha 2 delta 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 alpha 2 and delta subunits, respectively. The peptide band corresponding to the alpha 1 subunit was not detected in this gel electrophoresis. However, the alpha 1 subunit without bound alpha 2 delta was selectively eluted from MCC-1 Sepharose with 1% Triton X-100. A 190 kDa band corresponding to the alpha 1 subunit was visualized by fluorography and by silver staining in the fraction eluted with Triton X-100. Electrophoretically, the amount of alpha 1 was smaller than that of the alpha 2 subunit in the purified sample obtained here.
Tokumaru
H
H
Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Anzai
K
K
Abe
T
T
Kirino
Y
Y
D000818:Animals; D001665:Binding Sites; D015220:Calcium Channels / Q000032:analysis* / Q000302:isolation & purification; D002846:Chromatography, Affinity; D004095:Dihydropyridines; D009206:Myocardium / Q000737:chemistry*; D011817:Rabbits; D012508:Sarcolemma / Q000737:chemistry; D013552:Swine
D015220:Calcium Channels; D004095:Dihydropyridines; C038806:1,4-dihydropyridine