Solubilization and hydrodynamic characterization of the dihydropyridine receptor from rat ventricular muscle.
The dihydropyridine receptor-calcium channel complex, prelabeled with (+)-[3H]PN200-110, was solubilized from rat heart membranes with a detergent mixture of digitonin and Triton X-100. The dissociation of (+)-[3H]PN200-110 was slow enough to permit the hydrodynamic characterization of the complex by means of sucrose gradient sedimentation and gel filtration. The hydrodynamic properties of the complex were determined in several detergents, including Tween 80, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS), and digitonin. S20,w values of 12.5, 15.4, and 21.0 S were obtained in sucrose gradients prepared in Tween 80, CHAPS, and digitonin, respectively. A Stokes radius of 86-87 A was obtained in each of the three detergents. Determination of the partial specific volume of the protein-detergent complex in each case revealed that the differences in S20,w values could be explained by the differences in the properties of the bound detergent species. Partial specific volumes of 0.796, 0.730, and 0.730 ml/g, corresponding to molecular weights of 595,000, 540,000, and 740,000 were obtained for the complex in Tween 80, CHAPS, and digitonin, respectively. This indicated that Tween 80 readily exchanged for the solubilizing mixture of digitonin and Triton X-100, whereas CHAPS did not. Detergent exchange with Tween 80 made it possible to determine the fractional contribution of the receptor protein to the molecular weight of the protein-detergent complex. The molecular weight of the dihydropyridine receptor-calcium channel complex was estimated to be 370,000. The protein-detergent complex was found to have a frictional coefficient of 1.39, consistent with a large transmembrane protein.
D000818:Animals; D015220:Calcium Channels; D002499:Centrifugation, Density Gradient; D002793:Cholic Acids / Q000494:pharmacology; D002850:Chromatography, Gel; D003903:Deuterium; D017666:Deuterium Oxide; D004072:Digitonin / Q000494:pharmacology; D008297:Male; D008970:Molecular Weight; D009206:Myocardium / Q000032:analysis*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D011136:Polysorbates / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D011978:Receptors, Nicotinic / Q000032:analysis / Q000302:isolation & purification*; D012995:Solubility; D014867:Water
D015220:Calcium Channels; D002793:Cholic Acids; D011136:Polysorbates; D011978:Receptors, Nicotinic; D014867:Water; D011092:Polyethylene Glycols; D017830:Octoxynol; D003903:Deuterium; D017666:Deuterium Oxide; D004072:Digitonin; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate