The 1,4-dihydropyridine receptor associated with the skeletal muscle voltage-dependent Ca2+ channel. Purification and subunit composition.
The dihydropyridine receptor associated with the voltage-dependent Ca2+ channel from rabbit skeletal muscle has been purified using the tritiated derivative of (+)-PN 200-110. The drug was used not only as a marker associated with the solubilized receptor but also in direct binding experiments performed after each purification step. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate solubilization of a microsomal preparation resulted in an extract with a specific binding activity of 10 pmol/mg of protein. A combination of chromatographic steps utilizing anion exchange, lectin affinity, and gel filtration resulted in an 80-fold purification to a specific binding activity of 800 pmol/mg of protein. The affinity of (+)-[3H]PN 200-110 for the solubilized receptor was only slightly altered after the purification procedure. The KD values were 0.7 and 1.8 nM on the starting material and the most purified fractions, respectively. The subunit composition of the dihydropyridine receptor was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was consistent with three polypeptides of Mr 142,000, 33,000, and 32,000. The last two small components were not covalently associated with the larger one. In spite of a careful investigation of the conditions which improved the stability of the dihydropyridine receptor, a partial denaturation could not be prevented during purification. This resulted in an underestimation of receptor purity when calculated from the maximal specific binding activity as compared to the enrichment in the three polypeptides observed after polyacrylamide gel electrophoresis. Finally, application of the same purification procedure to solubilized microsomal preparations of chick and frog skeletal muscle demonstrated the presence of a large polypeptide component of Mr 135,000-141,000 associated with the Ca2+ channel from these sources. The doublet of small molecular weight was not found with the frog muscle.
D000818:Animals; D001001:Anura; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D002462:Cell Membrane / Q000032:analysis; D002645:Chickens; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004095:Dihydropyridines; D004591:Electrophoresis, Polyacrylamide Gel; D017275:Isradipine; D008861:Microsomes / Q000032:analysis; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis*; D010069:Oxadiazoles / Q000378:metabolism; D011725:Pyridines / Q000378:metabolism*; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D002121:Calcium Channel Blockers; D015220:Calcium Channels; D004095:Dihydropyridines; D010069:Oxadiazoles; D011725:Pyridines; D011978:Receptors, Nicotinic; C038806:1,4-dihydropyridine; D017275:Isradipine