Purification and affinity labeling of dihydropyridine receptor from rabbit skeletal muscle membranes.
Undegraded dihydropyridine (DHP)-receptor (putatively a voltage-gated Ca2+ channel) has been purified as a 340-kDa protein complex to approximately 80% homogeneity (2.4 nmol of DHP-receptor per mg of protein) from rabbit skeletal muscle by a rapid purification protocol. Transverse-tubule membranes were prepared in high yield by Ribi-press treatment. The DHP-receptor complex was solubilized in 1% digitonin followed by a two-step chromatographic purification procedure. The equilibrium dissociation constant of [3H](+)-PN200-110 binding (Kd; 0.9 nM) was not significantly changed by solubilization or purification. The purified DHP-receptor is composed of two subunits with apparent molecular masses of 148 kDa and 195 kDa migrating in polyacrylamide gels under nonreducing conditions as a single moiety of approximately 300 kDa. The 195-kDa subunit was affinity-labeled with [3H]azidopine in both transverse-tubule membranes and purified DHP-receptor preparations. The subunit can be degraded by high-energy irradiation to a 26-kDa peptide and by proteolysis to a 32-kDa peptide. Thus, it is probably due to proteolytic cleavage and/or photolysis that neither purification nor affinity-labeling studies have previously identified a DHP-receptor subunit of comparable molecular mass (195 kDa).
Kanngiesser
U
U
Lehrstuhl für Biochemie, Ruhr-Universität Bochum, Bochum-Querenburg, Federal Republic of Germany.
Nalik
P
P
Pongs
O
O
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D015220:Calcium Channels; D002499:Centrifugation, Density Gradient; D004095:Dihydropyridines / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D008566:Membranes / Q000032:analysis; D008970:Molecular Weight; D009132:Muscles / Q000032:analysis*; D010777:Photochemistry; D011817:Rabbits; D011978:Receptors, Nicotinic / Q000302:isolation & purification* / Q000378:metabolism
D000345:Affinity Labels; D015220:Calcium Channels; D004095:Dihydropyridines; D046911:Macromolecular Substances; D011978:Receptors, Nicotinic; C038806:1,4-dihydropyridine