Purification and characterization of the dihydropyridine-sensitive voltage-dependent calcium channel from cardiac tissue.
The dihydropyridine-sensitive voltage-dependent Ca2+ channel from cardiac tissue was purified 900-fold using DEAE-Sephadex A-25, concanavalin A-Sepharose, and wheat germ agglutinin-Sepharose. The purified preparation was highly enriched in a peptide of 140,000 daltons when electrophoresed on sodium dodecyl sulfate gels in the presence of 2-mercaptoethanol, or 170,000 when electrophoresed in the presence of iodoacetamide. Polyclonal antibodies raised against the purified subunits of the rabbit skeletal muscle Ca2+ channel recognized the 170-kDa protein in preparations electrophoresed under nonreducing conditions, and the large peptide of 140 kDa and smaller peptides of 29-32 kDa in preparations analyzed under reducing conditions. Monoclonal antibodies, which were raised against the native Ca2+ channel from skeletal muscle, immunoprecipitated [3H]PN 200-110 binding activity from solubilized cardiac membranes and immunoprecipitated 125I-labeled peptides (from the purified cardiac Ca2+ channel preparation) which migrated as a single species of 170 kDa under nonreducing conditions, or as 140, 32, and 29 kDa under reducing conditions. The results show that the purified cardiac Ca2+ channel, like that previously purified from skeletal muscle, consists of a major component of 170 kDa which is comprised of a 140-kDa peptide linked by disulfide bonds to smaller peptides of 32-29 kDa. Peptide maps of the 140-kDa peptide purified from cardiac and skeletal muscle preparations were strikingly similar, suggesting a high degree of homology in their primary sequence.
D000818:Animals; D000936:Antigen-Antibody Complex; D030161:Avian Proteins; D002118:Calcium / Q000378:metabolism; D002645:Chickens; D004095:Dihydropyridines; D007106:Immune Sera; D007460:Iodoacetamide / Q000494:pharmacology; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000302:isolation & purification* / Q000378:metabolism; D008623:Mercaptoethanol / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism*; D011725:Pyridines / Q000494:pharmacology*
D000936:Antigen-Antibody Complex; D030161:Avian Proteins; C051235:CCC protein, chicken; D004095:Dihydropyridines; D007106:Immune Sera; D007473:Ion Channels; D008565:Membrane Proteins; D011725:Pyridines; D008623:Mercaptoethanol; C038806:1,4-dihydropyridine; D002118:Calcium; D007460:Iodoacetamide