A rapid procedure for the purification of cardiac 1,4-dihydropyridine receptors from porcine heart.
Highly purified porcine cardiac sarcolemma was used as a source for purification of mammalian cardiac 1,4-dihydropyridine receptors associated with the voltage-dependent Ca2+ channel. The cardiac digitonin-solubilized receptor prelabeled with (+)-[3H]PN 200-110 was enriched at least 236-fold using an improved, rapid three-step purification protocol which could be completed within 12 h. The purity of the preparation was at least 22%, the yield of the receptors 24%. Photoaffinity labeling experiments with (-)-[3H]azidopine allowed the identification of the cardiac alpha 1 subunit. In contrast to the purified rabbit or guinea-pig skeletal muscle Ca2+ channel complex, none of the purified polypeptides underwent rapid and substantial phosphorylation by the catalytic subunit of the cyclic AMP-dependent protein kinase in vitro.
Haase
H
H
Zentralinstitut für Herz-Kreislaufforschung, Akademie der Wissenschaften, Berlin, F.R.G.
Striessnig
J
J
Holtzhauer
M
M
Vetter
R
R
Glossmann
H
H
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D002121:Calcium Channel Blockers; D015220:Calcium Channels / Q000378:metabolism; D004095:Dihydropyridines; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D017275:Isradipine; D008566:Membranes / Q000378:metabolism; D009206:Myocardium / Q000737:chemistry*; D010766:Phosphorylation; D010777:Photochemistry; D011494:Protein Kinases / Q000378:metabolism; D011978:Receptors, Nicotinic / Q000302:isolation & purification*; D012508:Sarcolemma / Q000378:metabolism; D013552:Swine
D000345:Affinity Labels; D001386:Azides; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D011978:Receptors, Nicotinic; C041130:azidopine; D011494:Protein Kinases; D017275:Isradipine